Engineered glucosyltransferases

ABSTRACT

Disclosed herein are glucosyltransferases with modified amino acid sequences. Such engineered enzymes exhibit improved alpha-glucan product yields and/or lower leucrose yields, for example. Further disclosed are reactions and methods in which engineered glucosyltransferases are used to produce alpha-glucan.

This application claims the benefit of U.S. Provisional Application No.62/557,840 (filed Sep. 13, 2017), which is incorporated herein byreference in its entirety.

FIELD

The present disclosure is in the field of enzyme catalysis. For example,the disclosure pertains to glucosyltransferase enzymes with modifiedamino acid sequences. Such modified enzymes have improved product yieldproperties, for example.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named20180911_CL6613USNP_SequenceListing_ST25 created on Sep. 11, 2018, andhaving a size of about 406 kilobytes and is filed concurrently with thespecification. The sequence listing contained in this ASCII-formatteddocument is part of the specification and is herein incorporated byreference in its entirety.

BACKGROUND

Driven by a desire to use polysaccharides in various applications,researchers have explored for polysaccharides that are biodegradable andthat can be made economically from renewably sourced feedstocks. Onesuch polysaccharide is alpha-1,3-glucan, an insoluble glucan polymercharacterized by having alpha-1,3-glycosidic linkages. This polymer hasbeen prepared, for example, using a glucosyltransferase enzyme isolatedfrom Streptococcus salivarius (Simpson et al., Microbiology141:1451-1460, 1995). Also for example, U.S. Pat. No. 7,000,000disclosed the preparation of a spun fiber from enzymatically producedalpha-1,3-glucan. Various other glucan materials have also been studiedfor developing new or enhanced applications. For example, U.S. PatentAppl. Publ. No. 2015/0232819 discloses enzymatic synthesis of severalinsoluble glucans having mixed alpha-1,3 and -1,6 linkages.

While these and other advances have been made in producing glucanpolymers using glucosyltransferase enzymes, less attention appears tohave been drawn to improving the glucan yields of such enzymes.Addressing this technological gap, disclosed herein areglucosyltransferases engineered to have modified amino acid sequencesendowing these enzymes with enhanced glucan production properties.

SUMMARY

In one embodiment, the present disclosure concerns a non-nativeglucosyltransferase comprising at least two amino acid substitutions atpositions corresponding with amino acid residues Gln-588, Phe-607, orArg-741 of SEQ ID NO:62, wherein the non-native glucosyltransferasesynthesizes alpha-glucan comprising 1,3-linkages, and wherein thenon-native glucosyltransferase has: (i) an alpha-glucan yield that ishigher than the alpha-glucan yield of a second glucosyltransferase thatonly differs from the non-native glucosyltransferase at the substitutionpositions, and/or (ii) a leucrose yield that is lower than the leucroseyield of the second glucosyltransferase.

In another embodiment, the present disclosure concerns a polynucleotidecomprising a nucleotide sequence encoding a non-nativeglucosyltransferase as presently disclosed, optionally wherein one ormore regulatory sequences are operably linked to the nucleotidesequence, and preferably wherein the one or more regulatory sequencesinclude a promoter sequence.

In another embodiment, the present disclosure concerns a reactioncomposition comprising water, sucrose, and a non-nativeglucosyltransferase as presently disclosed.

In another embodiment, the present disclosure concerns a method ofproducing alpha-glucan comprising: (a) contacting at least water,sucrose, and a non-native glucosyltransferase enzyme as presentlydisclosed, whereby alpha-glucan is produced; and b) optionally,isolating the alpha-glucan produced in step (a).

In another embodiment, the present disclosure concerns a method ofpreparing a polynucleotide sequence encoding a non-nativeglucosyltransferase, the method comprising: (a) identifying apolynucleotide sequence encoding a parent glucosyltransferase that (i)comprises an amino acid sequence that is at least about 40% identical toSEQ ID NO:4 or positions 55-960 of SEQ ID NO:4, and (ii) synthesizesalpha-glucan comprising 1,3-linkages; and (b) modifying thepolynucleotide sequence identified in step (a) to substitute at leasttwo amino acids of the parent glucosyltransferase at positionscorresponding with amino acid residues Gln-588, Phe-607 or Arg-741 ofSEQ ID NO:62, thereby providing a polynucleotide sequence encoding anon-native glucosyltransferase that has: (i) an alpha-glucan yield thatis higher than the alpha-glucan yield of the parent glucosyltransferase,and/or (ii) a leucrose yield that is lower than the leucrose yield ofthe parent glucosyltransferase.

BRIEF DESCRIPTION OF THE SEQUENCES

TABLE 1 Summary of Nucleic Acid and Protein SEQ ID Numbers^(b) Nucleicacid Protein Description SEQ ID NO. SEQ ID NO. GTF 0874, Streptococcussobrinus. The first 156 amino acids  1^(a)  2 of the protein are deletedcompared to GENBANK (1435 aa) Identification No. 450874; a startmethionine is included. GTF 6855, Streptococcus salivarius SK126. Thefirst 178  3^(a)  4 amino acids of the protein are deleted compared to(1341 aa) GENBANK Identification No. 228476855 (Acc. No. ZP_04061500.1);a start methionine is included. GTF 2379, Streptococcus salivarius. Thefirst 203 amino  5^(a)  6 acids of the protein are deleted compared toGENBANK (1247 aa) Identification No. 662379; a start methionine isincluded. GTF 7527 or GTFJ, Streptococcus salivarius. The first 42 7^(a)  8 amino acids of the protein are deleted compared to (1477 aa)GENBANK Identification No. 47527; a start methionine is included. GTF1724, Streptococcus downei. The first 162 amino acids  9^(a) 10 of theprotein are deleted compared to GENBANK (1436 aa) Identification No.121724; a start methionine is included. GTF 0544, Streptococcus mutans.The first 164 amino acids 11^(a) 12 of the protein are deleted comparedto GENBANK (1313 aa) Identification No. 290580544; a start methionine isincluded. GTF 5926, Streptococcus dentirousetti. The first 144 amino13^(a) 14 acids of the protein are deleted compared to GENBANK (1323 aa)Identification No. 167735926; a start methionine is included. GTF 4297,Streptococcus oralis. The first 228 amino acids of 15^(a) 16 the proteinare deleted compared to GENBANK Identification (1348 aa) No. 7684297; astart methionine is included. GTF 5618, Streptococcus sanguinis. Thefirst 223 amino 17^(a) 18 acids of the protein are deleted compared toGENBANK (1348 aa) Identification No. 328945618; a start methionine isincluded. GTF 2765, unknown Streptococcus sp. C150. The first 193 19^(a)20 amino acids of the protein are deleted compared to (1340 aa) GENBANKIdentification No. 322372765; a start methionine is included. GTF 0427,Streptococcus sobrinus. The first 156 amino acids 25^(a) 26 of theprotein are deleted compared to GENBANK (1435 aa) Identification No.940427; a start methionine is included. GTF 2919, Streptococcussalivarius PS4. The first 92 amino 27^(a) 28 acids of the protein aredeleted compared to GENBANK (1340 aa) Identification No. 383282919; astart methionine is included. GTF 2678, Streptococcus salivarius K12.The first 188 amino 29^(a) 30 acids of the protein are deleted comparedto GENBANK (1341 aa) Identification No. 400182678; a start methionine isincluded. GTF 3929, Streptococcus salivarius JIM8777. The first 17833^(a) 34 amino acids of the protein are deleted compared to (1341 aa)GENBANK Identification No. 387783929; a start methionine is included.GTF 3298, Streptococcus sp. C150. The first 209 amino 59 acids of theprotein are deleted compared to GENBANK (1242 aa) Identification No.322373298; a start methionine is included. Wild type GTFJ, Streptococcussalivarius. GENBANK 60 Identification No. 47527. (1518 aa) Wild type GTFcorresponding to GTF 2678, Streptococcus 61 salivarius K12. (1528 aa)Wild type GTF corresponding to GTF 6855, Streptococcus 62 salivariusSK126. (1518 aa) Wild type GTF corresponding to GTF 2919, Streptococcus63 salivarius PS4. (1431 aa) Wild type GTF corresponding to GTF 2765,unknown 64 Streptococcus sp. C150. (1532 aa) Shorter version of GTF7527, Streptococcus salivarius, (also 65 referred to as “7527-NT”herein. The first 178 amino acids of (1341 aa) the protein are deletedcompared to GENBANK Identification No. 47527; a start methionine isincluded. Terminator sequence added to pHY300PLK to derive the 67 pHYTvector. Aclglu1 alpha-glucosidase. 68  (971 aa) Nfiglu1alpha-glucosidase. 69  (969 aa) Ncrglu1 alpha-glucosidase. 70 (1022 aa)TauSec098_b alpha-glucosidase. 71 (1012 aa) TauSec098_calpha-glucosidase. 72  (984 aa) TauSec098_d alpha-glucosidase. 73  (984aa) TauSec099 alpha-glucosidase. 74  (973 aa) BloGlu1 alpha-glucosidase.75  (604 aa) BloGlu2 alpha-glucosidase. 76  (604 aa) BloGlu3alpha-glucosidase. 77  (604 aa) BpsGlu1 alpha-glucosidase. 78  (585 aa)BthGlu1 alpha-glucosidase. 79  (601 aa) BbrGlu2 alpha-glucosidase. 80 (662 aa) BbrGlu5 alpha-glucosidase. 81  (606 aa) ^(a)This DNA codingsequence is codon-optimized for expression in E. coli and is merelydisclosed as an example of a suitable coding sequence. ^(b)SEQ ID NOs:21-24, 31, 32, 35-58 and 66 are intentionally not included in this tableand merely serve as placeholders.

DETAILED DESCRIPTION

The disclosures of all cited patent and non-patent literature areincorporated herein by reference in their entirety.

Unless otherwise disclosed, the terms “a” and “an” as used herein areintended to encompass one or more (i.e., at least one) of a referencedfeature.

Where present, all ranges are inclusive and combinable, except asotherwise noted. For example, when a range of “1 to 5” is recited, therecited range should be construed as including ranges “1 to 4”, “1 to3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, and the like.

The terms “alpha-glucan”, “alpha-glucan polymer” and the like are usedinterchangeably herein. An alpha-glucan is a polymer comprising glucosemonomeric units linked together by alpha-glycosidic linkages. In typicalembodiments, an alpha-glucan herein comprises at least about 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% alpha-glycosidic linkages. Examples ofalpha-glucan polymers herein include alpha-1,3-glucan.

The terms “poly alpha-1,3-glucan”, “alpha-1,3-glucan”, “alpha-1,3-glucanpolymer” and the like are used interchangeably herein. Alpha-1,3-glucanis a polymer comprising glucose monomeric units linked together byglycosidic linkages, typically wherein at least about 50% of theglycosidic linkages are alpha-1,3. Alpha-1,3-glucan in certainembodiments comprises at least 90% or 95% alpha-1,3 glycosidic linkages.Most or all of the other linkages in alpha-1,3-glucan herein typicallyare alpha-1,6, though some linkages may also be alpha-1,2 and/oralpha-1,4.

The terms “glycosidic linkage”, “glycosidic bond”, “linkage” and thelike are used interchangeably herein and refer to the covalent bond thatjoins a carbohydrate (sugar) molecule to another group such as anothercarbohydrate. The term “alpha-1,3-glycosidic linkage” as used hereinrefers to the type of covalent bond that joins alpha-D-glucose moleculesto each other through carbons 1 and 3 on adjacent alpha-D-glucose rings.The term “alpha-1,6-glycosidic linkage” as used herein refers to thecovalent bond that joins alpha-D-glucose molecules to each other throughcarbons 1 and 6 on adjacent alpha-D-glucose rings. The glycosidiclinkages of a glucan polymer herein can also be referred to as“glucosidic linkages”. Herein, “alpha-D-glucose” will be referred to as“glucose”.

The glycosidic linkage profile of an alpha-glucan herein can bedetermined using any method known in the art. For example, a linkageprofile can be determined using methods using nuclear magnetic resonance(NMR) spectroscopy (e.g., ¹³C NMR or ¹H NMR). These and other methodsthat can be used are disclosed in, for example, Food Carbohydrates:Chemistry, Physical Properties, and Applications (S. W. Cui, Ed.,Chapter 3, S. W. Cui, Structural Analysis of Polysaccharides, Taylor &Francis Group LLC, Boca Raton, Fla., 2005), which is incorporated hereinby reference.

The “molecular weight” of large alpha-glucan polymers herein can berepresented as weight-average molecular weight (Mw) or number-averagemolecular weight (Mn), the units of which are in Daltons or grams/mole.Alternatively, the molecular weight of large alpha-glucan polymers canbe represented as DP_(w) (weight average degree of polymerization) orDP_(n) (number average degree of polymerization). The molecular weightof smaller alpha-glucan polymers such as oligosaccharides typically canbe provided as “DP” (degree of polymerization), which simply refers tothe number of glucoses comprised within the alpha-glucan. Various meansare known in the art for calculating these various molecular weightmeasurements such as with high-pressure liquid chromatography (HPLC),size exclusion chromatography (SEC), or gel permeation chromatography(GPC).

The term “sucrose” herein refers to a non-reducing disaccharide composedof an alpha-D-glucose molecule and a beta-D-fructose molecule linked byan alpha-1,2-glycosidic bond. Sucrose is known commonly as table sugar.

The terms “leucrose” and “D-glucopyranosyl-alpha(1-5)-D-fructopyranose”are used interchangeably herein and refer to a disaccharide containingan alpha-1,5 glucosyl-fructose linkage.

The terms “glucosyltransferase”, “glucosyltransferase enzyme”, “GTF”,“glucansucrase” and the like are used interchangeably herein. Theactivity of a glucosyltransferase herein catalyzes the reaction of thesubstrate sucrose to make the products alpha-glucan and fructose. Otherproducts (by-products) of a GTF reaction can include glucose, varioussoluble gluco-oligosaccharides, and leucrose. Wild type forms ofglucosyltransferase enzymes generally contain (in the N-terminal toC-terminal direction) a signal peptide (which is typically removed bycleavage processes), a variable domain, a catalytic domain, and aglucan-binding domain. A glucosyltransferase herein is classified underthe glycoside hydrolase family 70 (GH70) according to the CAZy(Carbohydrate-Active EnZymes) database (Cantarel et al., Nucleic AcidsRes. 37:D233-238, 2009).

The term “glucosyltransferase catalytic domain” herein refers to thedomain of a glucosyltransferase enzyme that providesalpha-glucan-synthesizing activity to a glucosyltransferase enzyme. Aglucosyltransferase catalytic domain typically does not require thepresence of any other domains to have this activity.

The terms “enzymatic reaction”, “glucosyltransferase reaction”, “glucansynthesis reaction”, “reaction composition”, “reaction formulation” andthe like are used interchangeably herein and generally refer to areaction that initially comprises water, sucrose, at least one activeglucosyltransferase enzyme, and optionally other components. Componentsthat can be further present in a glucosyltransferase reaction typicallyafter it has commenced include fructose, glucose, leucrose, solublegluco-oligosaccharides (e.g., DP2-DP7) (such may be considered asproducts or by-products, depending on the glucosyltransferase used),and/or insoluble alpha-glucan product(s) of DP8 or higher (e.g., DP100and higher). It would be understood that certain glucan products, suchas alpha-1,3-glucan with a degree of polymerization (DP) of at least 8or 9, are water-insoluble and thus not dissolved in a glucan synthesisreaction, but rather may be present out of solution (e.g., by virtue ofhaving precipitated from the reaction). It is in a glucan synthesisreaction where the step of contacting water, sucrose and aglucosyltransferase enzyme is performed. The term “under suitablereaction conditions” as used herein refers to reaction conditions thatsupport conversion of sucrose to alpha-glucan product(s) viaglucosyltransferase enzyme activity.

The “yield” of an alpha-glucan product in a glucosyltransferase reactionin some aspects herein represents the molar yield based on the convertedsucrose. The molar yield of an alpha-glucan product can be calculatedbased on the moles of the alpha-glucan product divided by the moles ofthe sucrose converted. Moles of converted sucrose can be calculated asfollows: (mass of initial sucrose−mass of final sucrose)/molecularweight of sucrose [342 g/mol]. This molar yield calculation can beconsidered as a measure of selectivity of the reaction toward thealpha-glucan. In some aspects, the “yield” of an alpha-glucan product ina glucosyltransferase reaction can be based on the glucosyl component ofthe reaction. Such a yield (yield based on glucosyl) can be measuredusing the following formula:

Alpha-Glucan Yield=((IS/2−(FS/2+LE/2+GL+SO))/(IS/2−FS/2))×100%.

The fructose balance of a glucosyltransferase reaction can be measuredto ensure that HPLC data, if applicable, are not out of range (90-110%is considered acceptable). Fructose balance can be measured using thefollowing formula:

Fructose Balance=((180/342×(FS+LE)+FR)/(180/342×IS))×100%.

In the above two formulae, IS is [Initial Sucrose], FS is [FinalSucrose], LE is [Leucrose], GL is [Glucose], SO is [Soluble Oligomers](gluco-oligosaccharides), and FR is [Fructose]; the concentrations ofeach foregoing substrate/product provided in double brackets are inunits of grams/L and as measured by HPLC, for example.

The terms “percent by volume”, “volume percent”, “vol %”, “v/v %” andthe like are used interchangeably herein. The percent by volume of asolute in a solution can be determined using the formula: [(volume ofsolute)/(volume of solution)]×100%.

The terms “percent by weight”, “weight percentage (wt %)”,“weight-weight percentage (% w/w)” and the like are used interchangeablyherein. Percent by weight refers to the percentage of a material on amass basis as it is comprised in a composition, mixture, or solution.

The terms “aqueous conditions”, “aqueous reaction conditions”, “aqueoussetting”, “aqueous system” and the like are used interchangeably herein.Aqueous conditions herein refer to a solution or mixture in which thesolvent is at least about 60 wt % water, for example. Aglucosyltransferase reaction herein is performed under aqueousconditions.

The terms “soluble”, “aqueous-soluble”, “water-soluble” and the like asused herein characterize a glucan that has the capability of dissolvingin water and/or an aqueous solution herein. Examples of soluble glucansherein are certain oligosaccharides, such as alpha-1,3-glucan with a DPless than 8. In contrast, a glucan that is “insoluble”,“aqueous-insoluble”, “water-insoluble” (and like terms) does notdissolve (or does not appreciably dissolve) in water and/or an aqueoussolution herein. Optionally, the conditions for determining solubilityinclude a water/solution temperature range of about 1 to 85° C. (e.g.,20-25° C.) and/or a pH range of about 4-9 (e.g., 6-8).

The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidmolecule” and the like are used interchangeably herein. These termsencompass nucleotide sequences and the like. A polynucleotide may be apolymer of DNA or RNA that is single- or double-stranded, thatoptionally contains synthetic, non-natural or altered nucleotide bases.A polynucleotide may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof.

The term “gene” as used herein refers to a DNA polynucleotide sequencethat expresses an RNA (RNA is transcribed from the DNA polynucleotidesequence) from a coding region, which RNA can be a messenger RNA(encoding a protein) or a non-protein-coding RNA. A gene may refer tothe coding region alone, or may include regulatory sequences upstreamand/or downstream to the coding region (e.g., promoters, 5′-untranslatedregions, 3′-transcription terminator regions). A coding region encodinga protein can alternatively be referred to herein as an “open readingframe” (ORF). A gene that is “native” or “endogenous” refers to a geneas found in nature with its own regulatory sequences; such a gene islocated in its natural location in the genome of a host cell. A“chimeric” gene refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature(i.e., the regulatory and coding regions are heterologous with eachother). Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orregulatory sequences and coding sequences derived from the same source,but arranged in a manner different than that found in nature. A“foreign” or “heterologous” gene can refer to a gene that is introducedinto the host organism by gene transfer. Foreign/heterologous genes cancomprise native genes inserted into a non-native organism, native genesintroduced into a new location within the native host, or chimericgenes. Polynucleotide sequences in certain embodiments disclosed hereinare heterologous. A “transgene” is a gene that has been introduced intothe genome by a gene delivery procedure (e.g., transformation). A“codon-optimized” open reading frame has its frequency of codon usagedesigned to mimic the frequency of preferred codon usage of the hostcell.

As used herein, the term “polypeptide” is defined as a chain of aminoacid residues, usually having a defined sequence. As used herein theterm polypeptide is interchangeable with the terms “peptides” and“proteins”. Typical amino acids contained in polypeptides herein include(respective three- and one-letter codes shown parenthetically): alanine(Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp,D), cysteine (Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q),glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu,L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F),proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp,W), tyrosine (Tyr, Y), valine (Val, V).

The term “heterologous” means not naturally found in the location ofinterest. For example, a heterologous gene can be one that is notnaturally found in a host organism, but that is introduced into the hostorganism by gene transfer. As another example, a nucleic acid moleculethat is present in a chimeric gene can be characterized as beingheterologous, as such a nucleic acid molecule is not naturallyassociated with the other segments of the chimeric gene (e.g., apromoter can be heterologous to a coding sequence).

A “non-native” amino acid sequence or polynucleotide sequence comprisedin a cell or organism herein does not occur in a native (natural)counterpart of such cell or organism. Such an amino acid sequence orpolynucleotide sequence can also be referred to as being heterologous tothe cell or organism.

“Regulatory sequences” as used herein refer to nucleotide sequenceslocated upstream of a gene's transcription start site (e.g., promoter),5′ untranslated regions, introns, and 3′ non-coding regions, and whichmay influence the transcription, processing or stability, and/ortranslation of an RNA transcribed from the gene. Regulatory sequencesherein may include promoters, enhancers, silencers, 5′ untranslatedleader sequences, introns, polyadenylation recognition sequences, RNAprocessing sites, effector binding sites, stem-loop structures, andother elements involved in regulation of gene expression. One or moreregulatory elements herein may be heterologous to a coding regionherein.

A “promoter” as used herein refers to a DNA sequence capable ofcontrolling the transcription of RNA from a gene. In general, a promotersequence is upstream of the transcription start site of a gene.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic DNA segments. Promoters that cause agene to be expressed in a cell at most times under all circumstances arecommonly referred to as “constitutive promoters”. A promoter mayalternatively be inducible. One or more promoters herein may beheterologous to a coding region herein.

A “strong promoter” as used herein refers to a promoter that can directa relatively large number of productive initiations per unit time,and/or is a promoter driving a higher level of gene transcription thanthe average transcription level of the genes in a cell.

The terms “3′ non-coding sequence”, “transcription terminator”,“terminator” and the like as used herein refer to DNA sequences locateddownstream of a coding sequence. This includes polyadenylationrecognition sequences and other sequences encoding regulatory signalscapable of affecting mRNA processing or gene expression.

As used herein, a first nucleic acid sequence is “hybridizable” to asecond nucleic acid sequence when a single-stranded form of the firstnucleic acid sequence can anneal to the second nucleic acid sequenceunder suitable annealing conditions (e.g., temperature, solution ionicstrength). Hybridization and washing conditions are well known andexemplified in Sambrook J, Fritsch E F and Maniatis T, MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory:Cold Spring Harbor, N.Y. (1989), which is incorporated herein byreference, particularly Chapter 11 and Table 11.1.

The term “DNA manipulation technique” refers to any technique in whichthe sequence of a DNA polynucleotide sequence is modified. Although theDNA polynucleotide sequence being modified can be used as a substrateitself for modification, it does not have to be physically in hand forcertain techniques (e.g., a sequence stored in a computer can be used asthe basis for the manipulation technique). A DNA manipulation techniquecan be used to delete and/or mutate one or more DNA sequences in alonger sequence. Examples of a DNA manipulation technique includerecombinant DNA techniques (restriction and ligation, molecularcloning), polymerase chain reaction (PCR), and synthetic DNA methods(e.g., oligonucleotide synthesis and ligation). Regarding synthetic DNAtechniques, a DNA manipulation technique can entail observing a DNApolynucleotide in silico, determining desired modifications (e.g., oneor more deletions) of the DNA polynucleotide, and synthesizing a DNApolynucleotide that contains the desired modifications.

The term “in silico” herein means in or on an information storage and/orprocessing device such as a computer; done or produced using computersoftware or simulation, i.e., virtual reality.

The terms “upstream” and “downstream” as used herein with respect topolynucleotides refer to “5′ of” and “3′ of”, respectively.

The term “expression” as used herein refers to (i) transcription of RNA(e.g., mRNA or a non-protein-coding RNA) from a coding region, and/or(ii) translation of a polypeptide from mRNA. Expression of a codingregion of a polynucleotide sequence can be up-regulated ordown-regulated in certain embodiments.

The term “operably linked” as used herein refers to the association oftwo or more nucleic acid sequences such that the function of one isaffected by the other. For example, a promoter is operably linked with acoding sequence when it is capable of affecting the expression of thatcoding sequence. That is, the coding sequence is under thetranscriptional control of the promoter. A coding sequence can beoperably linked to one (e.g., promoter) or more (e.g., promoter andterminator) regulatory sequences, for example.

The term “recombinant” when used herein to characterize a DNA sequencesuch as a plasmid, vector, or construct refers to an artificialcombination of two otherwise separated segments of sequence, e.g., bychemical synthesis and/or by manipulation of isolated segments ofnucleic acids by genetic engineering techniques.

The term “transformation” as used herein refers to the transfer of anucleic acid molecule into a host organism or host cell by any method. Anucleic acid molecule that has been transformed into an organism/cellmay be one that replicates autonomously in the organism/cell, or thatintegrates into the genome of the organism/cell, or that existstransiently in the cell without replicating or integrating. Non-limitingexamples of nucleic acid molecules suitable for transformation aredisclosed herein, such as plasmids and linear DNA molecules. Hostorganisms/cells herein containing a transforming nucleic acid sequencecan be referred to as “transgenic”, “recombinant”, “transformed”,“engineered”, as a “transformant”, and/or as being “modified forexogenous gene expression”, for example.

The terms “sequence identity”, “identity” and the like as used hereinwith respect to polynucleotide or polypeptide sequences refer to thenucleic acid residues or amino acid residues in two sequences that arethe same when aligned for maximum correspondence over a specifiedcomparison window. Thus, “percentage of sequence identity”, “percentidentity” and the like refer to the value determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the results by 100 to yield the percentage of sequenceidentity. It would be understood that, when calculating sequenceidentity between a DNA sequence and an RNA sequence, T residues of theDNA sequence align with, and can be considered “identical” with, Uresidues of the RNA sequence. For purposes of determining “percentcomplementarity” of first and second polynucleotides, one can obtainthis by determining (i) the percent identity between the firstpolynucleotide and the complement sequence of the second polynucleotide(or vice versa), for example, and/or (ii) the percentage of basesbetween the first and second polynucleotides that would create canonicalWatson and Crick base pairs.

Percent identity can be readily determined by any known method,including but not limited to those described in: 1) ComputationalMolecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2)Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.)Academic: NY (1993); 3) (Griffin, A. M., and Griffin, H. G., Eds.)Humana: NJ (1994); 4) Sequence Analysis in Molecular Biology (vonHeinje, G., Ed.) Academic (1987); and 5) Sequence Analysis Primer(Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991), all of whichare incorporated herein by reference.

Preferred methods for determining percent identity are designed to givethe best match between the sequences tested. Methods of determiningidentity and similarity are codified in publicly available computerprograms, for example. Sequence alignments and percent identitycalculations can be performed using the MEGALIGN program of theLASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.),for example. Multiple alignment of sequences can be performed, forexample, using the Clustal method of alignment which encompasses severalvarieties of the algorithm including the Clustal V method of alignment(described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in theMEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite(DNASTAR Inc.). For multiple alignments, the default values cancorrespond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Defaultparameters for pairwise alignments and calculation of percent identityof protein sequences using the Clustal method can be KTUPLE=1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids, theseparameters can be KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALSSAVED=4. Additionally, the Clustal W method of alignment can be used(described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci. 8:189-191(1992); Thompson, J. D. et al,Nucleic Acids Research, 22 (22): 4673-4680, 1994) and found in theMEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite(DNASTAR Inc.). Default parameters for multiple alignment(protein/nucleic acid) can be: GAP PENALTY=10/15, GAP LENGTHPENALTY=0.2/6.66, Delay Divergent Seqs (%)=30/30, DNA TransitionWeight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB.

Various polypeptide amino acid sequences and polynucleotide sequencesare disclosed herein as features of certain embodiments. Variants ofthese sequences that are at least about 70-85%, 85-90%, or 90%-95%identical to the sequences disclosed herein can be used or referenced.Alternatively, a variant amino acid sequence or polynucleotide sequencecan have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity with a sequence disclosed herein. Thevariant amino acid sequence or polynucleotide sequence has the samefunction/activity of the disclosed sequence, or at least about 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% of the function/activity of the disclosedsequence. Any polypeptide amino acid sequence disclosed herein notbeginning with a methionine can typically further comprise at least astart-methionine at the N-terminus of the amino acid sequence. Incontrast, any polypeptide amino acid sequence disclosed herein beginningwith a methionine can optionally lack such a methionine residue.

The terms “aligns with”, “corresponds with”, and the like can be usedinterchangeably herein. Some embodiments herein relate to aglucosyltransferase comprising at least one amino acid substitution at aposition corresponding with at least one particular amino acid residueof SEQ ID NO:62. An amino acid position of a glucosyltransferase orsubsequence thereof (e.g., catalytic domain or catalytic domain plusglucan-binding domains) (can refer to such an amino acid position orsequence as a “query” position or sequence) can be characterized tocorrespond with a particular amino acid residue of SEQ ID NO:62 (canrefer to such an amino acid position or sequence as a “subject” positionor sequence) if (1) the query sequence can be aligned with the subjectsequence (e.g., where an alignment indicates that the query sequence andthe subject sequence [or a subsequence of the subject sequence] are atleast about 30%, 40%, 50%, 60%, 70%, 80%, or 90% identical), and (2) ifthe query amino acid position directly aligns with (directly lines upagainst) the subject amino acid position in the alignment of (1). Ingeneral, one can align a query amino acid sequence with a subjectsequence (SEQ ID NO:62 or a subsequence of SEQ ID NO:62) using anyalignment algorithm, tool and/or software described disclosed herein(e.g., BLASTP, ClustalW, ClustalV, Clustal-Omega, EMBOSS) to determinepercent identity. Just for further example, one can align a querysequence with a subject sequence herein using the Needleman-Wunschalgorithm (Needleman and Wunsch, J. Mol. Biol. 48:443-453, 1970) asimplemented in the Needle program of the European Molecular Biology OpenSoftware Suite (EMBOSS [e.g., version 5.0.0 or later], Rice et al.,Trends Genet. 16:276-277, 2000). The parameters of such an EMBOSSalignment can comprise, for example: gap open penalty of 10, gapextension penalty of 0.5, EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix.

The numbering of particular amino acid residues of SEQ ID NO:62 herein(e.g., Leu-373, Leu-428, Ala-472, Ala-510, Leu-513, Met-529, Gln-588,Phe-607, Asn-613, Gln-616, Ser-631, Gly-633, Phe-634, Ser-710, Thr-635,Arg-722, Arg-741, Thr-877, Asp-948, Phe-951, Gln-957, Val-1188,Met-1253) is with respect to the full-length amino acid sequence of SEQID NO:62. The first amino acid (i.e., position 1, Met-1) of SEQ ID NO:62is at the start of the signal peptide. Unless otherwise disclosed,substitutions herein are with respect to the full-length amino acidsequence of SEQ ID NO:62.

A “non-native glucosyltransferase” herein (“mutant”, “variant”,“modified” and like terms can likewise be used to describe such aglucosyltransferase) has at least one amino acid substitution at aposition corresponding with a particular amino acid residue of SEQ IDNO:62. Such at least one amino acid substitution typically is in placeof the amino acid residue(s) that normally (natively) occurs at the sameposition in the native counterpart (parent) of the non-nativeglucosyltransferase (i.e., although SEQ ID NO:62 is used as a referencefor position, an amino acid substitution herein is with respect to thenative counterpart of a non-native glucosyltransferase) (consideredanother way, when aligning the sequence of a non-nativeglucosyltransferase with SEQ ID NO:62, determining whether asubstitution exists at a particular position does not dependin-and-of-itself on the respective amino acid residue in SEQ ID NO:62,but rather depends on what amino acid exists at the subject positionwithin the native counterpart of the non-native glucosyltransferase).The amino acid normally occurring at the relevant site in the nativecounterpart glucosyltransferase often (but not always) is the same as(or conserved with) the particular amino acid residue of SEQ ID NO:62for which the alignment is made. A non-native glucosyltransferaseoptionally can have other amino acid changes (mutations, deletions,and/or insertions) relative to its native counterpart sequence.

It may be instructive to illustrate a substitution/alignment herein. SEQID NO:12 (GTF 0544) is a truncated form of a Streptococcus sobrinusglucosyltransferase. It is noted that Leu-193 of SEQ ID NO:12corresponds with Leu-373 of SEQ ID NO:62 (alignment not shown). If SEQID NO:12 is mutated at position 193 to substitute the Leu residue with adifferent residue (e.g., Gln), then it can be stated that the position193-mutated version of SEQ ID NO:12 represents a non-nativeglucosyltransferase having an amino acid substitution at a positioncorresponding with Leu-373 of SEQ ID NO:62, for example.

The term “isolated” means a substance (or process) in a form orenvironment that does not occur in nature. Non-limiting examples ofisolated substances include (1) any non-naturally occurring substance(e.g., a non-native glucosyltransferase herein), (2) any substanceincluding, but not limited to, any host cell, enzyme, variant, nucleicacid, protein, peptide, cofactor, or carbohydrate/saccharide that is atleast partially removed from one or more or all of the naturallyoccurring constituents with which it is associated in nature; (3) anysubstance modified by the hand of man relative to that substance foundin nature (e.g., a non-native glucosyltransferase herein); or (4) anysubstance modified by increasing the amount of the substance relative toother components with which it is naturally associated. It is believedthat the embodiments (e.g., enzymes and reaction compositions) disclosedherein are synthetic/man-made, and/or have properties that are notnaturally occurring.

The term “increased” as used herein can refer to a quantity or activitythat is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 50%, 100%, or 200% morethan the quantity or activity for which the increased quantity oractivity is being compared. The terms “increased”, “elevated”,“enhanced”, “greater than”, “improved” and the like are usedinterchangeably herein. These terms can be used to characterize the“over-expression” or “up-regulation” of a polynucleotide encoding aprotein, for example.

While advances have been made in producing glucan polymers usingglucosyltransferase enzymes, less attention appears to have been drawnto improving the glucan yields of such enzymes. Addressing thistechnological gap, disclosed herein are glucosyltransferases engineeredto have modified amino acid sequences endowing these enzymes withenhanced glucan production properties.

Certain embodiments of the present disclosure concern a non-nativeglucosyltransferase comprising at least two or three amino acidsubstitutions at positions corresponding with amino acid residuesGln-588, Phe-607, and/or Arg-741 of SEQ ID NO:62, wherein the non-nativeglucosyltransferase synthesizes alpha-glucan comprising 1,3-linkages,and wherein the non-native glucosyltransferase has:

(i) an alpha-glucan yield that is higher than the alpha-glucan yield ofa second glucosyltransferase that only differs from the non-nativeglucosyltransferase at the substitution position(s), and/or

(ii) a leucrose yield that is lower than the leucrose yield of thesecond glucosyltransferase.

Thus, in general, mutant glucosyltransferase enzymes are disclosedherein that can synthesize higher amounts of alpha-glucan, and/or loweryields of leucrose, which is a by-product often considered undesirablewhen the main goal is alpha-glucan synthesis.

A non-native glucosyltransferase herein synthesizes alpha-glucancomprising 1,3-linkages. In some aspects, at least about 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 69%, 70%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% ofthe glycosidic linkages of such an alpha-glucan can be alpha-1,3linkages. The linkage profile of an alpha-glucan can optionally becharacterized as having a range between any two of these values. Theother linkages in any of these aspects having 30%-99% alpha-1,3 linkagescan be alpha-1,6, and/or not include any alpha-1,4 or alpha-1,2linkages, for example.

Alpha-glucan in some aspects can have, for example, less than 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% of alpha-1,2 or alpha-1,4glycosidic linkages. In another embodiment, an alpha-glucan only hasalpha-1,3 and optionally alpha-1,6 linkages (i.e., no alpha-1,2 oralpha-1,4 linkages).

Alpha-glucan in some aspects can be linear/unbranched (no branchpoints). Alternatively, there can be branches in an alpha-glucan herein.For example, an alpha-glucan can have less than about 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or 1% branch points as a percent of the linkages inthe polymer.

In certain aspects, an alpha-glucan can have a molecular weight inDP_(w) or DP_(n) of at least about 100. For example, the DP_(w) orDP_(n) can be about, or at least about, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100,or 1200. The molecular weight of an alpha-glucan can optionally beexpressed as a range between any two of these values (e.g., 100-1200,400-1200, 700-1200, 100-1000, 400-1000, 700-1000).

An alpha-glucan produced by a non-native glucosyltransferase hereintypically is water-insoluble. Alpha-1,3-glucan is generally insoluble ata DP_(w) of 8 or 9 and above in neutral (e.g., pH 6-8) aqueousconditions.

Any of the foregoing linkage profiles and/or molecular weight profiles,for example, can be combined herein to appropriately characterize analpha-glucan product of a non-native glucosyltransferase of the presentdisclosure. In some aspects, the linkage and/or molecular weight profileof an alpha-glucan product can be as disclosed in any of the followingpublications, all of which are incorporated herein by reference: U.S.Pat. Nos. 7,000,000 and 8,871,474, U.S. Patent Appl. Publ. No.2015/0232819.

A non-native glucosyltransferase, for example, can comprise the aminoacid sequence of any glucosyltransferase disclosed in the followingpublications that is capable of producing alpha-glucan as presentlydisclosed, but with the exception that the non-nativeglucosyltransferase comprises at least two of, or all three of, aminoacid substitutions at positions corresponding with amino acid residuesGln-588, Phe-607, and/or Arg-741 of SEQ ID NO:62: U.S. Pat. Nos.7,000,000 and 8,871,474; and U.S. Patent Appl. Publ. Nos. 2015/0232819and 2017/0002335, all of which are incorporated herein by reference. Insome aspects, such a non-native glucosyltransferase (i) comprises theforegoing substitutions, and (ii) comprises an amino acid sequence thatis at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or99.5% identical to the amino acid sequence of the respectivecounterpart/parent glucosyltransferase not having the foregoingsubstitutions.

In some aspects, a non-native glucosyltransferase (i) comprises at leasttwo of, or all three of, amino acid substitutions at positionscorresponding with amino acid residues Gln-588, Phe-607, and/or Arg-741of SEQ ID NO:62, and (ii) comprises or consists of an amino acidsequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 99.5% identical to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16,18, 20, 26, 28, 30, 34, or 59. Certain information regardingalpha-glucan products of glucosyltransferases with most of these aminoacid sequences is provided in Table 2.

TABLE 2 GTF Enzymes and Related Alpha-Glucan Products^(a) Linkages GTFSEQ ID Reducing Insoluble % alpha- % alpha- ID NO. Sugars Product 1,31,6 DP_(n) 0874 2 yes yes 100 0 60 6855 4 yes yes 100 0 440 2379 6 yesyes 37 63 310 7527 8 yes yes 100 0 440 1724 10 yes yes 100 0 250 0544 12yes yes 62 36 980 5926 14 yes yes 100 0 260 4297 16 yes yes 31 67 8005618 18 yes yes 34 66 1020 2765 20 yes yes 100 0 280 0427 26 yes yes 1000 120 2919 28 yes yes 100 0 250 2678 30 yes yes 100 0 390 3929 34 yesyes 100 0 280 ^(a)GTF reactions and product analyses were performed asfollows. Reactions were prepared comprising sucrose (50 g/L), potassiumphosphate buffer (pH 6.5, 20 mM) and a GTF enzyme (2.5% bacterial cellextract by volume; extracts prepared according to U.S. application Pub.No. 2017/0002335, in a manner similar to procedure disclosed in U.S.Pat. No. 8,871,474). After 24-30 hours at 22-25° C., insoluble productwas harvested by centrifugation, washed three times with water, washedonce with ethanol, and dried at 50° C. for 24-30 hours. Approximatelinkages and DP_(n) are shown for each insoluble product. Linkages andDP_(n) were determined by ¹³C NMR and SEC, respectively.

In some aspects, a non-native glucosyltransferase (i) comprises at leasttwo of, or all three of, amino acid substitutions at positionscorresponding with amino acid residues Gln-588, Phe-607 and/or Arg-741of SEQ ID NO:62, and (ii) comprises or consists of a glucosyltransferasecatalytic domain that is at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 99.5% identical to amino acid residues 55-960 ofSEQ ID NO:4, residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ IDNO:30, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ IDNO:20. Such a non-native glucosyltransferase, for instance, is believedto be able to produce alpha-glucan that is water-insoluble and compriseat least about 50% (e.g., 90% or 95%) alpha-1,3 linkages, and optionallyfurther have a DP_(w) of at least 100. It is noted that aglucosyltransferase with amino acid positions 54-957 of SEQ ID NO:65 canproduce alpha-1,3-glucan with 100% alpha-1,3 linkages and a DP_(w) of atleast 400 (data not shown, refer to Table 6 of U.S. Pat. Appl. Publ. No.2017/0002335, which is incorporated herein by reference), for example.It is further noted that SEQ ID NOs:65 (GTF 7527), 30 (GTF 2678), 4 (GTF6855), 28 (GTF 2919), and 20 (GTF 2765) each represent aglucosyltransferase that, compared to its respective wild typecounterpart, lacks the signal peptide domain and all or a substantialportion of the variable domain. Thus, each of these glucosyltransferaseenzymes has a catalytic domain followed by a glucan-binding domain. Theapproximate location of catalytic domain sequences in these enzymes isas follows: 7527 (residues 54-957 of SEQ ID NO:65), 2678 (residues55-960 of SEQ ID NO:30), 6855 (residues 55-960 of SEQ ID NO:4), 2919(residues 55-960 of SEQ ID NO:28), 2765 (residues 55-960 of SEQ IDNO:20). The amino acid sequences of the catalytic domains (approx.) ofGTFs 2678, 6855, 2919 and 2765 have about 94.9%, 99.0%, 95.5% and 96.4%identity, respectively, with the approximate catalytic domain sequenceof GTF 7527 (i.e., amino acids 54-957 of SEQ ID NO:65). Each of theseparticular glucosyltransferases (GTFs 2678, 6855, 2919 and 2765) canproduce alpha-1,3-glucan with 100% alpha-1,3 linkages and a DP_(w) of atleast 400 (data not shown, refer to Table 4 of U.S. Pat. Appl. Publ. No.2017/0002335). Based on this activity, and the relatedness (high percentidentity) of the foregoing catalytic domains, it is contemplated that anon-native glucosyltransferase herein having one of the foregoingcatalytic domains further with an amino acid substitution combination aspresently disclosed can produce alpha-glucan comprising at least about50% (e.g., 90% or 95%) alpha-1,3 linkages and a DP_(w) of at least 100.

In some aspects, a non-native glucosyltransferase (i) comprises at leasttwo of, or all three of, amino acid substitutions at positionscorresponding with amino acid residues Gln-588, Phe-607 and/or Arg-741of SEQ ID NO:62, and (ii) comprises or consists of an amino acidsequence that is at least about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 69%, 70%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical toSEQ ID NO:62 or a subsequence thereof such as SEQ ID NO:4 (without startmethionine thereof) or positions 55-960 of SEQ ID NO:4 (approximatecatalytic domain).

Although it is believed that a non-native glucosyltransferase in certainaspects need only have a catalytic domain, the non-nativeglucosyltransferase can be comprised within a larger amino acidsequence. For example, a catalytic domain may be linked at itsC-terminus to a glucan-binding domain, and/or linked at its N-terminusto a variable domain and/or signal peptide.

Although amino acid substitutions in a non-native glucosyltransferaseare generally disclosed herein with respect to the correspondingpositions in SEQ ID NO:62, such substitutions can alternatively bestated simply with respect to its position number in the sequence of thenon-native glucosyltransferase itself, as convenience may dictate.

Still further examples of non-native glucosyltransferases can be any asdisclosed herein and that include 1-300 (or any integer there between[e.g., 10, 15, 20, 25, 30, 35, 40, 45, or 50]) residues on theN-terminus and/or C-terminus. Such additional residues may be from acorresponding wild type sequence from which the glucosyltransferaseenzyme is derived, or may be a heterologous sequence such as an epitopetag (at either N- or C-terminus) or a heterologous signal peptide (atN-terminus), for example. A non-native glucosyltransferase hereintypically lacks an N-terminal signal peptide; such an enzyme canoptionally be characterized as being mature if its signal peptide wasremoved during a secretion process.

A non-native glucosyltransferase herein can be derived from anymicrobial source, for example, such as bacteria. Examples of bacterialglucosyltransferases are those derived from a Streptococcus species,Leuconostoc species, or Lactobacillus species. Examples of Streptococcusspecies include S. salivarius, S. sobrinus, S. dentirousetti, S. downei,S. mutans, S. oralis, S. gallolyticus and S. sanguinis. Examples ofLeuconostoc species include L. mesenteroides, L. amelibiosum, L.argentinum, L. carnosum, L. citreum, L. cremoris, L. dextranicum and L.fructosum. Examples of Lactobacillus species include L. acidophilus, L.delbrueckii, L. helveticus, L. salivarius, L. casei, L. curvatus, L.plantarum, L. sakei, L. brevis, L. buchneri, L. fermentum and L.reuteri.

A non-native glucosyltransferase herein can be prepared by fermentationof an appropriately engineered microbial strain, for example.Recombinant enzyme production by fermentation is well known in the artusing microbial species such as E. coli, Bacillus strains (e.g., B.subtilis), Ralstonia eutropha, Pseudomonas fluorescens, Saccharomycescerevisiae, Pichia pastoris, Hansenula polymorpha, and species ofAspergillus (e.g., A. awamori) and Trichoderma (e.g., T. reesei) (e.g.,see Adrio and Demain, Biomolecules 4:117-139, 2014, which isincorporated herein by reference). A nucleotide sequence encoding anon-native glucosyltransferase amino acid sequence is typically linkedto a heterologous promoter sequence to create an expression cassette forthe enzyme, and/or is codon-optimized accordingly. Such an expressioncassette may be incorporated in a suitable plasmid or integrated intothe microbial host chromosome, using methods well known in the art. Theexpression cassette may include a transcriptional terminator nucleotidesequence following the amino acid coding sequence. The expressioncassette may also include, between the promoter sequence andglucosyltransferase amino acid coding sequence, a nucleotide sequenceencoding a signal peptide (e.g., heterologous signal peptide) that isdesigned for direct secretion of the glucosyltransferase enzyme. At theend of fermentation, cells may be ruptured accordingly (generally when asignal peptide for secretion is not employed) and theglucosyltransferase enzyme can be isolated using methods such asprecipitation, filtration, and/or concentration. Alternatively, a lysateor extract comprising a glucosyltransferase can be used without furtherisolation. If the glucosyltransferase was secreted (i.e., it is presentin the fermentation broth), it can optionally be used as isolated from,or as comprised in, the fermentation broth. The activity of aglucosyltransferase enzyme can be confirmed by biochemical assay, suchas measuring its conversion of sucrose to glucan polymer.

A non-native glucosyltransferase herein can comprise amino acidsubstitutions at positions corresponding with at least two of, or allthree of, amino acid residues Gln-588, Phe-607 and/or Arg-741 of SEQ IDNO:62. In some aspects, the amino acid substitution at a positioncorresponding with amino acid Gln-588 of SEQ ID NO:62 can be with a Leu,Ala, or Val residue. In some aspects, the amino acid substitution at aposition corresponding with amino acid Phe-607 of SEQ ID NO:62 can bewith a Trp, Tyr, or Asn residue. In some aspects, the amino acidsubstitution at a position corresponding with amino acid Arg-741 of SEQID NO:62 can be with a Ser or Thr residue. Examples of a non-nativeglucosyltransferase herein comprise at least: (A) Gln-588-Ala,Phe-607-Tyr and Arg-741-Ser substitutions; (B) Gln-588-Leu, Phe-607-Trpand Arg-741-Ser substitutions; or (C) Gln-588-Leu, Phe-607-Tyr andArg-741-Thr substitutions. In some aspects, a non-nativeglucosyltransferase herein can comprise amino acid substitutions atpositions corresponding with amino acid residues (i) Gln-588 andPhe-607, (ii) Gln-588 and Arg-741, or (iii) Phe-607 and Arg-741 of SEQID NO:62.

A non-native glucosyltransferase herein can comprise, in addition to theforegoing two or three amino acid substitutions, one, two, three, four,five, six, seven, eight, nine, or more of the disclosed amino acidsubstitutions, for instance. For example, a non-nativeglucosyltransferase can further comprise at least one amino acidsubstitution at a position corresponding with amino acid residue Ala-510and/or Asp-948 of SEQ ID NO:62. In some aspects, the amino acidsubstitution at a position corresponding with amino acid Ala-510 of SEQID NO:62 can be with an Asp, Glu, Ile, or Val residue. In some aspects,the amino acid substitution at a position corresponding with amino acidAsp-948 of SEQ ID NO:62 can be with a Gly, Val, or Ala residue. Examplesof a non-native glucosyltransferase herein comprise at least: (D)Ala-510-Glu and Asp-948-Val substitutions; (E) an Asp-948-Alasubstitution; or (F) Ala-510-Asp and Asp-948-Gly substitutions, (inaddition to any of the foregoing substitution combinations of A, B, orC, for example).

In another example, a non-native glucosyltransferase can furthercomprise at least one amino acid substitution at a positioncorresponding with amino acid residue Ser-631, Ser-710, Arg-722, and/orThr-877 of SEQ ID NO:62. In some aspects, the amino acid substitution ata position corresponding with amino acid Ser-631 of SEQ ID NO:62 can bewith a Thr, Asp, Glu, or Arg residue. In some aspects, the amino acidsubstitution at a position corresponding with amino acid Ser-710 of SEQID NO:62 can be with a Gly, Ala, or Val residue. In some aspects, theamino acid substitution at a position corresponding with amino acidArg-722 of SEQ ID NO:62 can be with a His or Lys residue. In someaspects, the amino acid substitution at a position corresponding withamino acid Thr-877 of SEQ ID NO:62 can be with a Lys, His, or Argresidue. Examples of a non-native glucosyltransferase herein comprise atleast: (G) Ser-631-Thr, Ser-710-Gly, Arg-722-His and Thr-877-Lyssubstitutions; (H) Ser-710-Ala, Arg-722-Lys and Thr-877-Lyssubstitutions; or (I) Ser-631-Ser, Ser-710-Gly, and Thr-877-Argsubstitutions, (in addition to any of the foregoing substitutioncombinations of [i] A, B, or C; or [ii] A, B, or C with D, E, or F).

In another example, a non-native glucosyltransferase can furthercomprise at least one amino acid substitution at a positioncorresponding with amino acid residue Val-1188, Met-1253, and/or Gln-957of SEQ ID NO:62. In some aspects, the amino acid substitution at aposition corresponding with amino acid Val-1188 of SEQ ID NO:62 can bewith a Glu or Asp residue. In some aspects, the amino acid substitutionat a position corresponding with amino acid Met-1253 of SEQ ID NO:62 canbe with an Ile, Leu, Ala, or Val residue. In some aspects, the aminoacid substitution at a position corresponding with amino acid Gln-957 ofSEQ ID NO:62 can be with a Pro residue. Examples of a non-nativeglucosyltransferase herein comprise at least: (J) a Val-1188-Aspsubstitution; (K) a Met-1253-Ile substitution; (L) Val-1188-Glu andMet-1253-Ile substitutions; (M) Val-1188-Glu, Met-1253-Ile andGln-957-Pro substitutions; or (N) a Val-1188-Glu substitution (inaddition to any of the foregoing substitution combinations of [i] A, B,or C; [ii] A, B, or C with D, E, or F; [iii] A, B, or C with G, H, or I;[iv] A, B, or C with one of D, E, or F, and one of G, H, or I).

Other suitable substitutions that can be in addition to those listedabove, for example, include those as listed in Table 3 in Example 1(below) that are associated with (i) a decrease in leucrose productionby at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, and/or (ii) an increase inglucan yield by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%,115%, 120%, 125%, 130%, 135%, 140%, 145%, or 150%. In some aspects,suitable additional substitutions include those as listed in Table 3 inExample 1 (below) that are associated with a decrease in glucoseproduction by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, or 80%. In some aspects, suitableadditional substitutions include those as listed in Table 3 in Example 1(below) that are associated with a decrease in gluco-oligosaccharide(oligomer) production by at least about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, or 65%. The foregoing substitutions aslisted in Table 3 are as they correspond with the listed residueposition number in SEQ ID NO:62. In some aspects, one or moresubstitutions are conserved or non-conserved substitutions; suchconservation (or not) can be, for instance, with respect to the aminoacid that occurs in the native glucosyltransferase from which thenon-native glucosyltransferase is derived.

Simply for illustration purposes, a non-native glucosyltransferaseherein can comprise a combination of amino acid substitutions atpositions as follows (i-xix), where each substitution positioncorresponds with the respective amino acid position number of SEQ IDNO:62:

(i) A510, Q588, F607, R741, D948, R722, T877, M1253 and K1277;

(ii) A510, Q588, F607, R741, D948, R722, T877, V1188, M1253 and Q957;

(iii) A510, Q588, F607, R741, D948, T877, V1188, M1253 and Q957;

(iv) A510, Q588, F607, R741, D948 and M1253;

(v) A510, Q588, F607, R741 and D948;

(vi) Q588, F607, R741 and D948;

(vii) A510, Q588, F607, R741, D948, N628, T635, T877, M1253, F929 andR1172;

(viii) A510, Q588, F607, R741, D948, S631, S710, R722, T877, V1188 andM1253;

(ix) A510, Q588, F607, R741, D948, S631, S710, R722, T877 and V1188;

(x) A510, Q588, F607, R741, D948, S631, S710, T877, V1188 and M1253;

(xi) A510, Q588, F607, R741 and D948;

(xii) A510, Q588, F607, R741, D948 and V1188;

(xiii) A510, Q588, F607, R741, D948, S631, S710 and V1188;

(xiv) A510, Q588, F607, R741, D948, S710, R722, T877 and M1253;

(xv) A510, Q588, F607, R741, D948, S631, R722, T877, V1188 and M1253;

(xvi) A510, Q588, F607, R741, D948, S631, T877, V1188 and M1253;

(xvii) A510, Q588, F607, R741, D948, S631 and V1188;

(xviii) A510, Q588, F607, R741, D948, S631, R722, T877, V1188 and M1253;or

(xix) A510, Q588, F607, R741, D948, V1188 and M1253,

Some particular examples of embodiments i-xix are disclosed in Example 4below (Table 7). Thus, a non-native glucosyltransferase in some aspectscan comprise one of the following combinations of substitutions(xx-xxxviii), where each substitution corresponds with the respectiveamino acid residue of SEQ ID NO:62:

(xx) A510D/Q588L/F607Y/R741S/D948G/R722H/T877K/M1253I/K1277N,

(xxi) A510D/Q588L/F607Y/R741S/D948G/R722H/T877K/V1188E/M1253I/Q957P,

(xxii) A510D/Q588L/F607Y/R741S/D948G/T877K/V1188E/M1253I/Q957P,

(xxiii) A510D/Q588L/F607Y/R741S/D948G/M1253I,

(xxiv) A510D/Q588L/F607W/R741S/D948G,

(xxv) Q588L/F607Y/R741S/D948G,

(xxvi)

A510D/Q588L/F607Y/R741S/D948G/N628D/T635A/T877K/M1253I/F929L/R11720,

(xxvii)

A510D/Q588L/F607W/R741S/D948G/S631T/S710G/R722H/T877K/V1188E/M1253I,

(xxviii) A510D/Q588L/F607W/R741S/D948G/S631T/S710G/R722H/T877K/V1188E,

(xxix) A510D/Q588L/F607W/R741S/D948G/S631T/S710G/T877KA/1188E/M1253I,

(xxx) A510D/Q588L/F607Y/R741S/D948G,

(xxxi) A510D/Q588L/F607Y/R741S/D948GA/1188E,

(xxxii) A510D/Q588L/F607W/R741S/D948G/S631T/S710G/V1188E,

(xxxxiii) A510D/Q588L/F607W/R741S/D948G/S710G/R722H/T877K/M1253I,

(xxxiv) A510D/Q588L/F607Y/R741S/D948G/S631T/R722H/T877K/V1188E/M1253I,

(xxxv) A510D/Q588L/F607W/R741S/D948G/S631T/T877K/V1188E/M1253I,

(xxxvi) A510D/Q588L/F607W/R741S/D948G/S631T/V1188E,

(xxxvii) A510D/Q588L/F607Y/R741S/D948G/S631T/R722H/T877K/V1188E/M1253I,or

(xxxviii) A510D/Q588L/F607W/R741S/D948G/V1188E/M1253I.

A non-native glucosyltransferase with a combination of amino acidsubstitutions herein can be based on any of a variety ofglucosyltransferase amino acid sequences as presently disclosed, forexample. Simply for illustration purposes, examples of such a non-nativeglucosyltransferase include those with a combination of amino acidsubstitutions as described herein (e.g., any of embodiments i-xxxviiiabove) and comprising or consisting of an amino acid sequence that is atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%identical to SEQ ID NO:65 (optionally without the start methionine ofSEQ ID NO:65) or residues 54-957 of SEQ ID NO:65, SEQ ID NO:30(optionally without the start methionine of SEQ ID NO:30) or residues55-960 of SEQ ID NO:30, SEQ ID NO:4 (optionally without the startmethionine of SEQ ID NO:4) or residues 55-960 of SEQ ID NO:4, SEQ IDNO:28 (optionally without the start methionine of SEQ ID NO:28) orresidues 55-960 of SEQ ID NO:28, or SEQ ID NO:20 (optionally without thestart methionine of SEQ ID NO:20) or residues 55-960 of SEQ ID NO:20.

A non-native glucosyltransferase herein can have (i) an alpha-glucanyield that is higher than the alpha-glucan yield of a secondglucosyltransferase (or, simply, “another” glucosyltransferase) (e.g.,parent glucosyltransferase) that only differs from the non-nativeglucosyltransferase at the substitution positions, and/or (ii) aleucrose yield that is lower than the leucrose yield of the secondglucosyltransferase. A second glucosyltransferase herein, for example,can be comprised of all of, or mostly, native amino acid sequence. Thus,while a second glucosyltransferase herein can be a nativeglucosyltransferase in some aspects, it can be a prior-modifiedglucosyltransferase in other aspects (e.g., a glucosyltransferase withone or more other amino acid substitutions differing from thesubstitution[s] of the present disclosure). In some embodiments, asecond glucosyltransferase to which a non-native glucosyltransferase iscompared has native amino acid residues at the substitution positions.Determining whether an amino acid residue is native can be done bycomparing the second glucosyltransferase amino acid sequence to thenative/wild type glucosyltransferase amino acid sequence from which thesecond glucosyltransferase is derived. Optionally, a non-nativeglucosyltransferase in some embodiments can be characterized as havinghigher selectivity toward alpha-glucan synthesis (as compared toby-product synthesis).

In some aspects, a non-native glucosyltransferase herein can have analpha-glucan yield that is at least about 5%, 10%, 20%, 40%, 60%, 80%,100%, 120%, 140%, 160%, 180%, 200%, 220%, 240%, 260%, 280%, 300%, 320%,340%, 360%, or 380% higher than the alpha-glucan yield of a secondglucosyltransferase as presently disclosed. In some additional oralternative embodiments, a non-native glucosyltransferase can have adecrease in leucrose yield by at least about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%compared to the leucrose yield of a second glucosyltransferase. Thesedeterminations (alpha-glucan and/or leucrose yield) can be made withrespect to any glucan synthesis reaction/process as disclosed herein(e.g., taking into account initial sucrose conc., temperature, pH,and/or reaction time), and using any suitable measurement technique(e.g., HPLC or NIR spectroscopy). Typically, a comparison betweennon-native and second glucosyltransferases herein can be made underidentical or similar reaction conditions. The yield of aglucosyltransferase reaction in some aspects can be measured based onthe glucosyl component of the reaction.

In some embodiments, a non-native glucosyltransferase can exhibit adecrease in the yield of soluble gluco-oligosaccharides by at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, or 75% compared to the soluble gluco-oligosaccharide yield of asecond glucosyltransferase. A soluble gluco-oligosaccharide in someaspects can be DP2-7 or DP2-8, and have any linkage profile disclosedherein. In some aspects, the DP is or up to 10, 15, 20, or 25, but witha linkage profile allowing solubility (e.g., not over 90% or 95%alpha-1,3).

In some embodiments, a non-native glucosyltransferase can exhibit adecrease in the yield of glucose by at least about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% comparedto the glucose yield of a second glucosyltransferase.

Some embodiments disclosed herein concern a polynucleotide comprising anucleotide sequence that encodes a non-native glucosyltransferase aspresently disclosed. Optionally, one or more regulatory sequences areoperably linked to the nucleotide sequence, and preferably a promotersequence is included as a regulatory sequence.

A polynucleotide comprising a nucleotide sequence encoding a non-nativeglucosyltransferase herein can be a vector or construct useful fortransferring a nucleotide sequence into a cell, for example. Examples ofa suitable vector/construct can be selected from a plasmid, yeastartificial chromosome (YAC), cosmid, phagemid, bacterial artificialchromosome (BAC), virus, or linear DNA (e.g., linear PCR product). Apolynucleotide sequence in some aspects can be capable of existingtransiently (i.e., not integrated into the genome) or stably (i.e.,integrated into the genome) in a cell. A polynucleotide sequence in someaspects can comprise, or lack, one or more suitable marker sequences(e.g., selection or phenotype marker).

A polynucleotide sequence in certain embodiments can comprise one ormore regulatory sequences operably linked to the nucleotide sequenceencoding a non-native glucosyltransferase. For example, a nucleotidesequence encoding a non-native glucosyltransferase may be in operablelinkage with a promoter sequence (e.g., a heterologous promoter). Apromoter sequence can be suitable for expression in a cell (e.g.,bacterial cell such as E. coli or Bacillus; eukaryotic cell such as afungus, yeast, insect, or mammalian cell) or in an in vitro proteinexpression system, for example. Examples of other suitable regulatorysequences include transcription terminator sequences.

Some aspects herein are drawn to a cell comprising a polynucleotidesequence as presently disclosed; such a cell can be any type disclosedherein (e.g., bacterial cell such as E. coli or Bacillus; eukaryoticcell such as a fungus, yeast, insect, or mammalian cell). A cell canoptionally express a non-native glucosyltransferase encoded by thepolynucleotide sequence. In some aspects, the polynucleotide sequenceexists transiently (i.e., not integrated into the genome) or stably(i.e., integrated into the genome) in the cell.

Some embodiments disclosed herein concern reaction compositionscomprising water, sucrose, and one or more non-nativeglucosyltransferases herein. Such a reaction composition produces, atleast, alpha-glucan comprising 1,3-linkages as disclosed.

The temperature of a reaction composition herein can be controlled, ifdesired, and can be about 5-50° C., 20-40° C., 30-40° C., 20-30° C.,20-25° C., 20° C., 25° C., 30° C., 35° C., or 40° C., for example.

The initial concentration of sucrose in a reaction composition hereincan be about 20-400 g/L, 75-175 g/L, or 50-150 g/L, for example. In someaspects, the initial sucrose concentration is at least about 50, 75,100, 150 or 200 g/L, or is about 50-600 g/L, 100-500 g/L, 50-100 g/L,100-200 g/L, 150-450 g/L, 200-450 g/L, or 250-600 g/L. “Initialconcentration of sucrose” refers to the sucrose concentration in areaction composition just after all the reaction components have beenadded/combined (e.g., at least water, sucrose, non-nativeglucosyltransferase enzyme).

The pH of a reaction composition in certain embodiments can be about4.0-9.0, 4.0-8.5, 4.0-8.0, 5.0-8.0, 5.5-7.5, or 5.5-6.5. In someaspects, the pH can be about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or8.0. The pH can be adjusted or controlled by the addition orincorporation of a suitable buffer, including but not limited to:phosphate, tris, citrate, or a combination thereof. The bufferconcentration in a reaction composition herein can be about 0.1-300 mM,0.1-100 mM, 10-100 mM, 10 mM, 20 mM, or 50 mM, for example.

A reaction composition can be contained within any vessel (e.g., aninert vessel/container) suitable for applying one or more of thereaction conditions disclosed herein. An inert vessel in some aspectscan be of stainless steel, plastic, or glass (or comprise two or more ofthese components) and be of a size suitable to contain a particularreaction. For example, the volume/capacity of an inert vessel (and/orthe volume of a reaction composition herein), can be about, or at leastabout, 1, 10, 50, 100, 500, 1000, 2500, 5000, 10000, 12500, 15000, or20000 liters. An inert vessel can optionally be equipped with a stirringdevice.

A reaction composition herein can contain one, two, or moreglucosyltransferase enzymes, for example, just as long that at least oneof the enzymes is a non-native glucosyltransferase as presentlydisclosed. In some embodiments, only one or two glucosyltransferaseenzymes is/are comprised in a reaction composition. Aglucosyltransferase reaction herein can be, and typically is, cell-free(e.g., no whole cells present).

Any of the features disclosed herein (e.g., above and in the belowExamples) regarding a reaction composition can characterize appropriateaspects of a glucan production method herein, and vice versa.

The present disclosure also concerns a method for producingalpha-glucan, the method comprising: (a) contacting at least water,sucrose, and at least one non-native glucosyltransferase as disclosedherein that produces an alpha-glucan, whereby alpha-glucan is produced;and b) optionally, isolating the alpha-glucan produced in step (a).Conducting such a method, which can optionally be characterized as aglucan synthesis method, is typically also performed when conducting areaction composition herein.

A glucan synthesis method as presently disclosed comprises contacting atleast water, sucrose, and a non-native glucosyltransferase herein thatproduces an alpha-glucan. These and optionally other reagents can beadded altogether or in any order as discussed below. This step canoptionally be characterized as providing a reaction compositioncomprising water, sucrose and a non-native glucosyltransferase enzymethat synthesizes alpha-glucan. The contacting step herein can beperformed in any number of ways. For example, the desired amount ofsucrose can first be dissolved in water (optionally, other componentsmay also be added at this stage of preparation, such as buffercomponents), followed by addition of glucosyltransferase enzyme. Thesolution may be kept still, or agitated via stirring or orbital shaking,for example. A glucan synthesis method can be performed by batch,fed-batch, continuous mode, or by any variation of these modes.

Completion of a reaction in certain embodiments can be determinedvisually (e.g., no more accumulation of insoluble glucan), and/or bymeasuring the amount of sucrose left in the solution (residual sucrose),where a percent sucrose consumption of at least about 90%, 95%, or 99%can indicate reaction completion. A reaction of the disclosed processcan be conducted for about 1 hour to about 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 36, 48, 60, 72, 96, 120, 144, or 168 hours, for example.

The yield of an alpha-glucan produced in some aspects of a glucansynthesis method herein can be at least about 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%. Yield insome aspects can be measured based on the glucosyl component of thereaction. In some additional or alternative embodiments, the yield ofleucrose can be less than about 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%,10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. Such a yield in alpha-glucanand/or leucrose in some aspects is achieved in a reaction conducted forabout 16-24 hours (e.g., ˜20 hours), and/or is as measured using HPLC orNIR spectroscopy.

Insoluble alpha-glucan produced in a method herein can optionally beisolated. In certain embodiments, isolating insoluble alpha-glucan caninclude at least conducting a step of centrifugation and/or filtration.Isolation can optionally further comprise washing alpha-glucan one, two,or more times with water or other aqueous liquid, and/or drying thealpha-glucan product.

An isolated alpha-glucan product herein, as provided in a dry form, cancomprise no more than 2.0, 1.5, 1.0, 0.5, 0.25, 0.10, 0.05, or 0.01 wt %water, for example. In some aspects, an alpha-glucan product is providedin an amount of at least 1 gram (e.g., at least about 2.5, 5, 10, 25,50, 100, 250, 500, 750, 1000, 2500, 5000, 7500, 10000, 25000, 50000, or100000 g); such an amount can be a dry amount, for example.

A glucan synthesis method in some aspects can further comprisecontacting a soluble fraction of the glucosyltransferase reaction,and/or the glucosyltransferase reaction itself, with analpha-glucosidase enzyme to hydrolyze at least one glycosidic linkage ofone or more oligosaccharides present in the soluble fraction and/orglucosyltransferase reaction, thereby increasing the monosaccharidecontent in the soluble fraction. A soluble fraction herein can becontacted with an alpha-glucosidase after its separation from aninsoluble fraction comprising alpha-1,3-glucan, or before its separation(e.g., while it is being formed in the reaction, and/or after completionof the reaction) (i.e., in contacting step [a] and/or after separationstep [b]). A soluble fraction can be a filtrate or supernatant, forexample, of a glucosyltransferase reaction, and is typically obtainedfollowing the completion of insoluble alpha-1,3-glucan synthesis.Examples of suitable alpha-glucosidases herein include those comprisingan amino acid sequence that (i) is 100% identical to, or at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% A identical to, anyof SEQ ID NOs:68-81, and (ii) has hydrolytic activity toward alpha-1,5glucosyl-fructose linkages, alpha-1,3 glucosyl-glucose linkages, and/oralpha-1,6 glucosyl-glucose linkages in saccharides.

Any of the disclosed conditions for synthesizing an alpha-glucan, suchas the foregoing or those described in the below Examples, can beapplied to practicing a reaction composition as presently disclosed (andvice versa), and/or used to characterize features/activity of anon-native glucosyltransferase, accordingly.

The present disclosure also concerns a method of preparing apolynucleotide sequence encoding a non-native glucosyltransferaseherein. This method comprises:

-   -   (a) identifying a polynucleotide sequence encoding a parent        glucosyltransferase that (i) comprises an amino acid sequence        that is at least about 40% identical to SEQ ID NO:4 or positions        55-960 of SEQ ID NO:4, and (ii) synthesizes alpha-glucan        comprising 1,3-linkages; and    -   (b) modifying the polynucleotide sequence identified in step (a)        to substitute at least two or three amino acids of the parent        glucosyltransferase at positions corresponding with amino acid        residues Gln-588, Phe-607 and/or Arg-741 of SEQ ID NO:62,        thereby providing a polynucleotide sequence encoding a        non-native glucosyltransferase that has:        -   (i) an alpha-glucan yield that is higher than the            alpha-glucan yield of the parent glucosyltransferase, and/or        -   (ii) a leucrose yield that is lower than the leucrose yield            of the parent glucosyltransferase.            Such a method can optionally further comprise using a            polynucleotide prepared in this manner in a method of            expressing the non-native glucosyltransferase encoded by the            polynucleotide. Such an expression method can follow any            heterologous protein expression method as known in the art,            for example. The present method of preparing a            polynucleotide can optionally alternatively be characterized            as a method of increasing the product yield of a            glucosyltransferase.

Identification step (a) herein can, in some instances, compriseidentifying an amino acid sequence of a parent glucosyltransferaseenzyme. A polynucleotide sequence could be determined from this aminoacid sequence according to the genetic code (codons), such as thegenetic code used in the species from which the parentglucosyltransferase was identified.

Identifying a polynucleotide encoding a parent glucosyltransferaseherein can be performed (a) in silico, (b) with a method comprising anucleic acid hybridization step, (c) with a method comprising a proteinsequencing step, and/or (d) with a method comprising a protein bindingstep, for example.

Regarding in silico detection, the amino acid sequences of candidateparent glucosyltransferase enzymes (and/or nucleotide sequences encodingsuch glucosyltransferase enzymes) stored in a computer or database(e.g., public databases such as GENBANK, EMBL, REFSEQ, GENEPEPT,SWISS-PROT, PIR, PDB) can be reviewed in silico to identify aglucosyltransferase enzyme comprising an amino acid sequence with apercent sequence identity as described above for a parentglucosyltransferase. Such review could comprise using any means known inthe art such as through use of an alignment algorithm or software asdescribed above (e.g., BLASTN, BLASTP, ClustalW, ClustalV,Clustal-Omega, EMBOSS).

Identifying a parent glucosyltransferase as disclosed above canoptionally be performed via a method comprising a nucleic acidhybridization step. Such a method can comprise using DNA hybridization(e.g., Southern blot, dot blot), RNA hybridization (e.g., northernblot), or any other method that has a nucleic acid hybridization step(e.g., DNA sequencing, PCR, RT-PCR, all of which may comprisehybridization of an oligonucleotide), for example. A polynucleotidesequence encoding SEQ ID NO:4 or a subsequence thereof (e.g., positions55-960 of SEQ ID NO:4) can be used as a probe, for example, in such ahybridization. Conditions and parameters for carrying out hybridizationmethods in general are well known and disclosed, for example, inSambrook J, Fritsch E F and Maniatis T, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989);Silhavy T J, Bennan M L and Enquist L W, Experiments with Gene Fusions,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); AusubelF M et al., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-Interscience, Hoboken, N.J. (1987); andInnis M A, Gelfand D H, Sninsky J J and White T J (Editors), PCRProtocols: A Guide to Methods and Applications, Academic Press, Inc.,San Diego, Calif. (1990).

Identifying a parent glucosyltransferase as disclosed above canoptionally be performed via a method comprising a protein sequencingstep. Such a protein sequencing step can comprise one or more proceduressuch as N-terminal amino acid analysis, C-terminal amino acid analysis,Edman degradation, or mass spectrometry, for example.

Identifying a parent glucosyltransferase as disclosed above canoptionally be performed via a method comprising a protein binding step.Such a protein binding step can be performed using an antibody thatbinds to a motif or epitope within SEQ ID NO:4 (e.g., within positions55-960 of SEQ ID NO:4), for example.

A polynucleotide identified in step (a) (i.e., before its modificationin step [b]) can, in some aspects, encode a glucosyltransferasecomprising an amino acid sequence that is identical to, or at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the aminoacid sequence of any glucosyltransferase disclosed in Table 1. Analpha-glucan as produced by such a glucosyltransferase can be asdisclosed herein, for example.

A method of preparing a polynucleotide sequence encoding a non-nativeglucosyltransferase herein comprises step (b) of modifying thepolynucleotide sequence (encoding a parent glucosyltransferase)identified in step (a). Such modification substitutes at least two orthree amino acids of the parent glucosyltransferase at positionscorresponding with amino acid residues Gln-588, Phe-607 and/or Arg-741of SEQ ID NO:62. The non-native glucosyltransferase (encoded by themodified polynucleotide sequence) resulting from such substitutions canbe optionally be characterized as a “child glucosyltransferase” herein.

A parent glucosyltransferase enzyme herein can comprise an amino acidsequence that is at least about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 69%, 70%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical toSEQ ID NO:4 (optionally without start methionine thereof) or positions55-960 of SEQ ID NO:4 (approximate catalytic domain), for example. It isnoted simply for reference purposes that SEQ ID NO:4 without its startmethionine is a subsequence of SEQ ID NO:62.

A suitable modification of a polynucleotide in step (b) can be madefollowing any DNA manipulation technique known in the art. Modifyingstep (b) can optionally be performed in silico, followed by synthesis ofthe polynucleotide sequence encoding a non-native glucosyltransferase.For example, a polynucleotide sequence identified in step (a) can bemanipulated in silico using a suitable sequence manipulationprogram/software (e.g., VECTOR NTI, Life Technologies, Carlsbad, Calif.;DNAStrider; DNASTAR, Madison, Wis.). Following such virtualmanipulation, the modified polynucleotide sequence can be artificiallysynthesized by any suitable technique (e.g., annealing-based connectionof oligonucleotides, or any technique disclosed in Hughes et al.,Methods Enzymol. 498:277-309, which is incorporated herein byreference). It should be appreciated that the foregoing methodology isnot believed to necessarily rely on having a pre-existing polynucleotide(encoding a parent glucosyltransferase) in hand.

Modifying step (b) can optionally be performed using a physical copy ofa polynucleotide sequence identified in step (a) encoding a parentglucosyltransferase. As an example, such a polynucleotide can serve as atemplate for amplification using primers designed in a manner such thatthe amplified product encodes a non-native glucosyltransferase herein(e.g., refer to Innis et al., ibid.).

The amino acid substitutions in this method can be any of thosecombinations of substitutions as disclosed herein. Essentially anynon-native glucosyltransferase as presently disclosed can be encoded bya polynucleotide as prepared by this method, for instance, andconsequently can have the higher alpha-glucan yield and/or lowerleucrose yield profiles disclosed herein.

Non-limiting examples of compositions and methods disclosed hereininclude:

1. A non-native glucosyltransferase comprising at least two or threeamino acid substitutions at positions corresponding with amino acidresidues Gln-588, Phe-607, and/or Arg-741 of SEQ ID NO:62, wherein thenon-native glucosyltransferase synthesizes alpha-glucan comprising1,3-linkages, and wherein the non-native glucosyltransferase has: (i) analpha-glucan yield that is higher than the alpha-glucan yield of asecond glucosyltransferase that only differs from the non-nativeglucosyltransferase at the substitution positions, and/or (ii) aleucrose yield that is lower than the leucrose yield of the secondglucosyltransferase.2. The non-native glucosyltransferase of embodiment 1, wherein theglucosyltransferase comprises amino acid substitutions at positionscorresponding with amino acid residues Gln-588, Phe-607 and Arg-741 ofSEQ ID NO:62.3. The non-native glucosyltransferase of embodiment 1 or 2, wherein: (i)the amino acid substitution at the position corresponding with aminoacid residue Gln-588 is with a Leu, Ala, or Val residue; (ii) the aminoacid substitution at the position corresponding with amino acid residuePhe-607 is with a Trp, Tyr, or Asn residue; and/or (iii) the amino acidsubstitution at the position corresponding with amino acid residueArg-741 is with a Ser or Thr residue.4. The non-native glucosyltransferase of embodiment 1, 2, or 3, whereinthe glucosyltransferase further comprises at least one amino acidsubstitution at a position corresponding with amino acid residue Ala-510and/or Asp-948 of SEQ ID NO:62; optionally wherein: (i) the amino acidsubstitution at the position corresponding with amino acid residueAla-510 is with an Asp, Glu, Ile, or Val residue; and/or (ii) the aminoacid substitution at the position corresponding with amino acid residueAsp-948 is with a Gly, Val, or Ala residue.5. The non-native glucosyltransferase of embodiment 1, 2, 3, or 4,wherein the glucosyltransferase further comprises at least one aminoacid substitution at a position corresponding with amino acid residueSer-631, Ser-710, Arg-722, and/or Thr-877 of SEQ ID NO:62; optionallywherein: (i) the amino acid substitution at the position correspondingwith amino acid residue Ser-631 is with a Thr, Asp, Glu, or Arg residue;(ii) the amino acid substitution at the position corresponding withamino acid residue Ser-710 is with a Gly, Ala, or Val residue; (iii) theamino acid substitution at the position corresponding with amino acidresidue Arg-722 is with a His or Lys residue; and/or (iv) the amino acidsubstitution at the position corresponding with amino acid residueThr-877 is with a Lys, His, or Arg residue.6. The non-native glucosyltransferase of embodiment 1, 2, 3, 4, or 5,wherein the glucosyltransferase further comprises at least one aminoacid substitution at a position corresponding with amino acid residueVal-1188, Met-1253, and/or Gln-957 of SEQ ID NO:62; optionally wherein:(i) the amino acid substitution at the position corresponding with aminoacid residue Val-1188 is with a Glu or Asp residue; (ii) the amino acidsubstitution at the position corresponding with amino acid residueMet-1253 is with an Ile, Leu, Ala, or Val residue; and/or (iii) theamino acid substitution at the position corresponding with amino acidresidue Gln-957 is with a Pro residue.7. The non-native glucosyltransferase of embodiment 1, 2, 3, 4, 5, or 6,wherein the alpha-glucan produced by the non-native glucosyltransferaseis insoluble and comprises at least about 50% alpha-1,3 linkages, andoptionally wherein it has a weight average degree of polymerization(DP_(w)) of at least 100.8. The non-native glucosyltransferase of embodiment 7, comprising acatalytic domain that is at least about 90% identical to residues 55-960of SEQ ID NO:4, residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQID NO:30, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ IDNO:20.9. The non-native glucosyltransferase of embodiment 7 or 8, comprisingan amino acid sequence that is at least about 90% identical to SEQ IDNO:4, SEQ ID NO:65, SEQ ID NO:30, SEQ ID NO:28, or SEQ ID NO:20.10. The non-native glucosyltransferase of embodiment 1, 2, 3, 4, 5, 6,7, 8, or 9, wherein the non-native glucosyltransferase synthesizesinsoluble alpha-1,3-glucan having at least about 90% (or at least 95%)alpha-1,3-linkages.11. The non-native glucosyltransferase of embodiment 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, wherein the alpha-glucan yield is at least about 10%higher than the alpha-glucan yield of the second glucosyltransferase.12. A polynucleotide comprising a nucleotide sequence encoding anon-native glucosyltransferase according to any one of embodiments 1-11,optionally wherein one or more regulatory sequences are operably linkedto the nucleotide sequence, and preferably wherein the one or moreregulatory sequences include a promoter sequence.13. A reaction composition comprising water, sucrose, and a non-nativeglucosyltransferase according to any one of embodiments 1-11.14. A method of producing alpha-glucan comprising: (a) contacting atleast water, sucrose, and a non-native glucosyltransferase enzymeaccording to any one of embodiments 1-11, whereby alpha-glucan isproduced; and (b) optionally, isolating the alpha-glucan produced instep (a).15. A method of preparing a polynucleotide sequence encoding anon-native glucosyltransferase (e.g., of any one of embodiments 1-11),the method comprising: (a) identifying a polynucleotide sequenceencoding a parent glucosyltransferase that (i) comprises an amino acidsequence that is at least about 40% identical to SEQ ID NO:4 orpositions 55-960 of SEQ ID NO:4, and (ii) synthesizes alpha-glucancomprising 1,3-linkages; and (b) modifying the polynucleotide sequenceidentified in step (a) to substitute at least two or three amino acidsof the parent glucosyltransferase at positions corresponding with aminoacid residues Gln-588, Phe-607, and/or Arg-741 of SEQ ID NO:62, therebyproviding a polynucleotide sequence encoding a non-nativeglucosyltransferase that has: (i) an alpha-glucan yield that is higherthan the alpha-glucan yield of the parent glucosyltransferase, and/or(ii) a leucrose yield that is lower than the leucrose yield of theparent glucosyltransferase.16. The method of embodiment 15, wherein the identifying step isperformed: (a) in silico, (b) with a method comprising a nucleic acidhybridization step, (c) with a method comprising a protein sequencingstep, and/or (d) with a method comprising a protein binding step; and/orwherein the modifying step is performed: (e) in silico, followed bysynthesis of the polynucleotide sequence encoding the non-nativeglucosyltransferase enzyme, or (f) using a physical copy of thepolynucleotide sequence encoding the parent glucosyltransferase.

EXAMPLES

The present disclosure is further exemplified in the following Examples.It should be understood that these Examples, while indicating certainpreferred aspects herein, are given by way of illustration only. Fromthe above discussion and these Examples, one skilled in the art canascertain the essential characteristics of the disclosed embodiments,and without departing from the spirit and scope thereof, can makevarious changes and modifications to adapt the disclosed embodiments tovarious uses and conditions.

Example 1 Analysis of Amino Acid Sites Affecting GlucosyltransferaseSelectivity Toward Alpha-Glucan Synthesis

This Example describes screening for glucosyltransferase variants withimproved selectivity toward alpha-glucan synthesis from sucrose. Anotheraim of this screening was to identify glucosyltransferase variants thatexhibit reduced synthesis of by-products such as leucrose andgluco-oligosaccharides. Variants having either or both of these yieldproperties were identified.

The amino acid sequence of the glucosyltransferase used to prepare aminoacid substitutions in this Example was SEQ ID NO:4 (GTF 6855), whichessentially is an N-terminally truncated (signal peptide and variableregion removed) version of the full-length wild type glucosyltransferase(represented by SEQ ID NO:62) from Streptococcus salivarius SK126 (seeTable 1). Substitutions made in SEQ ID NO:4 can be characterized assubstituting for native amino acid residues, as each amino acidresidue/position of SEQ ID NO:4 (apart from the Met-1 residue of SEQ IDNO:4) corresponds accordingly with an amino acid residue/position withinSEQ ID NO:62. In reactions comprising at least sucrose and water, theglucosyltransferase of SEQ ID NO:4 typically produces alpha-glucanhaving about 100% alpha-1,3 linkages and a DP_(w) of 400 or greater(e.g., refer to U.S. Pat. Nos. 8,871,474 and 9,169,506, and U.S. Pat.Appl. Publ. No. 2017/0002336, which are incorporated herein byreference). This alpha-glucan product, which is insoluble, can beisolated following enzymatic synthesis via filtration, for example.

To summarize this Example, GTF 6855 variants (each with a single aminoacid substitution) from site evaluation libraries (SEL) were eachbacterially expressed, purified, and normalized to a concentration of100 ppm. Each enzyme preparation was then screened (in triplicate) usingsucrose as substrate in alpha-1,3 glucan synthesis reactions. Inaddition to determining the amount of alpha-1,3 glucan polymer producedin each reaction, the soluble sugar products (fructose, glucose,leucrose, gluco-oligosaccharides) and residual sucrose of each reactionwere analyzed by HPLC after about a 20-hour incubation.

Plasmids for individually expressing various single aminoacid-substituted variants of GTF 6855 (SEQ ID NO:4) in a Bacillussubtilis host were prepared. Such plasmids were prepared as follows. ADNA expression cassette having (operably linked in 5′-to-3′ order) theB. subtilis aprE promoter, a codon-optimized sequence encoding SEQ IDNO:4 (GTF 6855), and a BPN′ terminator was synthesized. This expressioncassette was cloned into the pHYT replicating shuttle vector (formingpHYT-GTF6855) and transformed into B. subtilis CBS12-1. The pHYT vectorwas derived from pHY300PLK (Takara) by adding a terminator sequence (SEQID NO:67) after the tetracycline resistance gene using the BstEII andEcoRI sites. The HindIII site in pHY300PLK had been removed by cloning alinker sequence (not shown) into the BamHI and HindIII sites. ThepHYT-GTF6855 plasmid was amplified and used for generating SELs. Theresulting plasmids encoding single-amino acid substituted GTFs weresequenced to verify each substitution.

To produce GTF 6855 (SEQ ID NO:4) and single amino acid-substitutedvariants thereof, B. subtilis individually transformed with pHYT-GTF6855or mutated versions thereof were cultivated in Tryptone Soya Broth(Oxoid Ltd., UK) and Grant's II medium. Heart infusion agar plates(Difco Laboratories, MI) were used to select transformants. Plasmidintegrity was maintained by the addition of 25 μg/mL tetracycline. EachGTF targeted for expression was detected in the growth medium afterincubation for about 6 hours at 37° C. After centrifugation andfiltration, culture supernatants with expressed GTF were obtained. GTFenzyme present in the supernatant was purified to apparent homogeneityby affinity chromatography using washed (2×MILLIQ 1×25 mM NaH₂PO₄ pH 5.7with intermediate centrifugation steps 100× g) SUPERDEX 200 resin (GEHealthcare). Each GTF was eluted with a 15% solution of Dextran T1(Pharmacosmos) in 25 mM NaH₂PO₄ pH 5.7 by centrifugation 100× g. Eachpurified GTF was dialyzed against 25 mM NaH₂PO₄ pH 5.7 buffer (at least100×) using a Harvard Apparatus 96-well DISPODIALYZER (10000-DaltonMWCO).

After dialysis, GTF enzyme concentration was determined by OD280 usingpurified GTF 6855 as a standard. Normalization of each purified GTF to100 ppm was achieved by diluting appropriately with 25 mM NaH₂PO₄ pH5.7. Protein concentration for each sample was confirmed using anAGILENT 1200 (Agilent Technologies) HPLC equipped with an AGILENT BIOSEC3 guard-column column (3 μm 100 Å (4.6×50 mm). Five (5) μL of samplewas injected onto the column for each determination. Compounds wereeluted with isocratic flow of 25 mM KH₂PO₄ pH 6.8+0.1 M NaCl for 1.3 minat 0.5 mL/min flow rate.

Each GTF (GTF 6855 and each variant thereof) was entered into a reactionwith sucrose to determine yield and selectivity. Each reaction wasperformed as follows: 37.5 μL of 100 ppm enzyme sample (ppm based on aBSA calibration curve) was added to 262.5 μL of 86 g/L sucrose (75 g/Lfinal) in 20 mM Na₂HPO₄/NaH₂PO₄ pH 5.7 and incubated overnight (about 20hours) at 30° C. After this incubation, each reaction was quenched byincubation for 1 hour at 80° C. A 200-4 aliquot of each quenchedreaction was filtered in vacuo via a 0.45-μm filter plate (Millipore0.45-μm Hydrophilic) and each filtrate was diluted 5× (10 μL sample+40μL 20 mM Na₂HPO₄/NaH₂PO₄) in preparation for HPLC sugar analysis.

Sucrose, glucose, fructose, leucrose and relative oligosaccharideconcentrations in each diluted filtrate were determined using an AGILENT1200 (Agilent Technologies) HPLC equipped with a 150×7.80 mm PHENOMENEXREZEX RNM carbohydrate Na⁺ 8% column PHENOMENX KRUDKATCHER 0.5-μm guardcolumn. The column was operated at 80° C. with an isocratic flow-rate of0.9 m L/min with 10 mM Na₂HPO₄/NaH₂PO₄ pH 6.7 (5 min per sample). FiveμL of diluted sample was injected. Appropriate sucrose, glucose,fructose, and leucrose calibration curves were used to determine sugarconcentrations. A mixture of purified gluco-oligosaccharides was used todetermine oligomer concentration.

The profiles of reactions (˜20 hours) as measured via the abovemethodology are provided in Table 3.

TABLE 3 Product Profiles of GTF 6855 (SEQ ID NO: 4) and Single AminoAcid-Substituted Variants thereof Alpha-1,3 Sucrose Leucrose GlucoseFructose Oligomers Glucan^(f) Fructose GTF (g/L)^(d) (g/L)^(d) (g/L)^(d)(g/L)^(d) (g/L)^(d,e) Yield^(i) Balance Plate 1^(a) 6855^(b) 1.6 21.16.3 28.9 9.1 31% 97% 6855^(b) 1.6 21.3 6.3 29.1 10.5 27% 98% 6855^(b)1.6 21.2 6.3 29.3 10.0 29% 98% 6855^(b) 1.6 21.1 6.3 28.9 10.8 27% 97%V186A^(c) 1.6 21.3 6.4 28.8 10.7 27% 97% V186M 1.6 21.4 6.4 28.7 10.627% 97% E194C 1.6 21.2 6.3 29.0 9.4 30% 98% L434N 1.9 22.7 7.1 28.4 12.718% 99% A472C 31.0 2.6 2.5 23.8 4.6 38% 99% A472S 5.3 2.8 13.9 36.5 9.131% 97% A510E 8.5 5.4 5.5 34.5 5.6 53% 100%  A510E 1.9 6.5 5.6 36.7 6.158% 98% A510I 4.3 6.8 5.4 35.2 5.4 57% 98% A510V 1.7 9.5 6.4 35.6 6.851% 99% L513Y 1.4 10.3 4.2 35.3 7.2 54% 99% M529L 1.9 10.4 4.2 35.2 10.944% 99% K578M 1.6 21.0 6.4 28.8 10.8 27% 97% Y605W 6.1 8.0 2.6 33.3 5.459% 97% F607N 8.4 11.4 4.1 30.5 7.1 45% 98% F607W 9.1 4.6 3.8 33.9 8.649% 98% N613I 4.5 7.7 6.4 35.8 14.8 29% 101%  N613M 2.7 11.0 5.3 34.612.1 37% 100%  N613T 1.7 10.3 4.6 35.0 7.1 53% 98% N613V 2.8 0.0 6.337.3 12.1 48% 92% Q616E 3.9 2.4 5.8 37.3 8.8 53% 97% K625A 1.5 21.2 6.329.4 9.9 29% 99% K625M 1.5 21.3 6.3 29.3 10.6 27% 99% S631T 5.4 11.4 4.632.0 7.6 46% 97% T635H 4.1 11.0 5.0 32.7 8.2 46% 97% T635W 13.1 8.5 4.529.6 7.0 42% 98% I636H 7.0 11.7 5.0 31.1 8.1 42% 98% D947G 2.4 19.1 6.129.8 9.9 31% 98% F951Y 4.0 1.5 9.9 38.0 15.4 28% 97% E849M 1.4 20.7 6.229.5 10.4 29% 98% Q1007A 1.4 19.4 6.2 30.2 10.1 31% 98% D1003G 13.8 10.74.6 28.3 5.4 42% 98% A1022M 1.7 20.6 6.2 29.3 12.2 24% 98% D1028L 1.622.1 6.6 28.9 11.6 23% 99% D1028Q 1.6 21.7 6.5 29.4 10.9 26% 99% A1057H1.5 21.4 6.4 29.2 10.6 27% 98% N1096A 1.6 22.4 6.6 28.6 10.7 25% 98%E1132A 1.5 21.4 6.4 29.2 10.6 27% 98% E1132H 1.5 21.3 6.4 29.2 10.5 27%98% E1132K 1.5 21.4 6.4 29.2 10.4 27% 98% E1132R 1.5 21.6 6.4 29.1 10.826% 99% L1212N 1.5 20.9 6.3 29.5 10.4 28% 98% T1431M 1.5 21.4 6.3 29.410.5 27% 99% A1442R 1.5 21.3 6.4 29.1 10.6 27% 98% Dead^(g) 79.4 0.0 0.00.0 0.0  0% 100%  Blank^(h) 79.7 0.0 0.1 0.0 0.0  0% 100%  Blank^(h)80.1 0.0 0.0 0.0 0.0  0% 100%  Plate 2^(a) 6855^(b) 1.4 20.1 6.4 28.210.0 29% 99% 6855^(b) 1.4 20.1 6.4 28.2 10.1 28% 99% 6855^(b) 1.4 20.06.3 28.3 10.3 28% 99% 6855^(b) 1.5 20.2 6.3 28.2 10.0 29% 100% Y219C^(c) 1.5 20.6 6.5 27.7 10.7 25% 99% E243H 1.4 20.3 6.3 28.2 10.128% 100%  L373A 2.4 11.3 11.2 27.4 21.6 −7% 87% L373Q 4.0 7.5 10.7 28.421.5 −2% 87% L373V 2.5 11.6 11.5 27.5 21.8 −9% 88% A377I 2.9 15.5 6.629.3 11.3 29% 98% D425Q 1.8 15.3 5.3 30.3 9.6 39% 99% L428V 5.3 10.5 6.230.8 8.2 42% 98% N475F 6.1 26.8 20.5 24.9 7.2 −16%  106%  N475W 1.5 61.87.5 9.1 1.9 −8% 106%  L513F 1.0 10.9 4.6 33.3 7.1 55% 99% L513W 1.3 11.54.9 32.4 8.9 48% 98% M529N 3.5 11.6 4.8 31.6 7.6 49% 99% I608Y 2.4 15.75.7 29.9 9.8 35% 99% N613G 2.2 10.5 5.0 33.5 10.6 43% 101%  N613L 2.913.3 5.0 32.1 11.7 35% 102%  D617E 8.4 10.2 6.9 29.8 9.0 34% 99% E621T1.5 18.6 6.0 29.1 10.4 30% 100%  I623H 69.8 0.2 1.4 3.3 0.0  4% 101% I627W 7.7 12.2 5.2 28.9 7.9 40% 99% S631D 9.8 12.3 5.7 27.5 8.0 35% 98%S631E 10.1 12.6 5.6 27.3 8.0 35% 99% S631R 6.7 12.3 5.4 28.7 8.1 40% 97%G633W 7.0 7.2 5.5 31.9 8.5 46% 99% F634A 7.4 8.4 5.7 30.8 8.2 43% 98%T635E 1.6 17.2 6.0 29.9 9.5 35% 100%  T635I 1.5 17.4 6.2 30.5 10.1 32%102%  T635Y 13.8 8.0 4.6 28.0 6.7 43% 99% A510E 2.5 5.9 5.5 34.8 4.3 66%99% N904E 5.7 6.9 12.6 32.5 13.5 15% 98% K930G 1.4 19.8 6.2 28.4 10.030% 99% K930V 1.4 19.6 6.3 28.6 10.0 30% 100%  D947F 1.4 20.3 6.2 27.89.9 29% 99% D947I 1.4 19.9 6.3 28.6 10.7 27% 100%  D947K 1.4 19.9 6.228.6 9.7 30% 100%  D947N 1.4 20.5 6.3 27.9 10.0 28% 99% D947Q 1.4 19.56.2 28.4 9.6 31% 99% D947S 1.3 18.9 6.1 28.8 9.4 33% 99% D947V 1.4 19.86.2 28.3 9.7 30% 99% D947Y 1.4 20.7 6.3 28.1 10.0 28% 100%  Q1007S 1.318.3 6.1 29.1 9.6 33% 99% D1003N 3.6 13.1 5.7 30.5 9.8 38% 99% I1026H1.4 19.4 6.2 28.7 9.7 31% 100%  D1028A 1.5 20.1 6.5 28.4 10.8 26% 100% D1028M 1.5 20.4 6.6 28.1 11.1 24% 100%  V1037A 1.5 20.2 6.4 28.4 10.328% 100%  K1041A 4.3 19.6 6.5 27.0 10.7 23% 99% K1041M 1.5 20.5 6.4 28.010.5 26% 100%  D1080M 1.4 20.0 6.4 28.3 10.1 29% 99% F1244P 1.4 19.6 6.328.6 9.9 30% 100%  F1244Q 1.4 19.7 6.4 28.6 9.9 30% 100%  T1431Q 1.420.0 6.2 28.5 8.9 33% 100%  G1484P 1.5 20.1 6.3 28.5 9.2 31% 100% W1437N 1.4 19.5 6.0 28.9 8.4 35% 100%  Dead^(g) 75.5 0.0 0.0 0.0 0.0  0%100%  Blank^(h) 75.5 0.0 0.0 0.0 0.0  0% 100%  Blank^(h) 76.0 0.0 0.00.0 0.5 −2% 100%  ^(a)Glucan synthesis reactions were run in microtiterplate format (two plates). ^(b)GTF 6855, SEQ ID NO: 4. Reactions withthis GTF were run in quadruplicate per plate. ^(c)Each listed GTF with asubstitution is a version of GTF 6855 comprising a substitution at arespective position, where the position number is in correspondence withthe residue numbering of SEQ ID NO: 62. The wild type residue is listedfirst (before residue position number) and the substituting residue islisted second (after the residue position number) (this “wild typeresidue-position number-variant residue” annotation format appliesthroughout the present disclosure). ^(d)Sucrose, leucrose, glucose,fructose and oligomers were measured as present in filtrate preparedpost reaction. ^(e)“Oligomers”, gluco-oligosaccharides (believed to allor mostly be of DP ≤ 7 or 8). ^(f)Insoluble alpha-1,3 glucan product.^(g)GTF with destroyed activity was entered into the reaction. ^(h)NoGTF was added to the reaction. ^(i)Alpha-glucan yield based on glucosyl.

Based on the data in Table 3, it is apparent that certain single aminoacid substitutions in GTF 6855 (SEQ ID NO:4) can increase this enzyme'syield of alpha-1,3-glucan and/or decrease its leucrose yield in glucansynthesis reactions, for example.

Example 2 Analysis of the Effects of Single Amino Acid Substitutions onOther Glucosyltransferases

This Example describes the effects of certain single amino acidsubstitutions on the activities of glucosyltransferases other than GTF6855 (SEQ ID NO:4). In general, it appears that substitutionscorresponding to (or similar to) those observed in Example 1 having asignificant effect on alpha-glucan and/or leucrose yields may be usefulfor imparting similar effects to different glucosyltransferases.

Phe-607-Tyr

Example 1 demonstrated, for example, that substitutions in GTF 6855 (SEQID NO:4) at the position corresponding to position 607 of SEQ ID NO:62affected enzyme activity (Table 3). In particular, substitutions of thePhe residue with an Asn or Trp residue both had significant effects onalpha-1,3 glucan yield (increased) and leucrose yield (decreased)compared to the respective yields of the non-substituted enzyme.

To test whether a similar substitution could similarly affect yields ina different GTF, a substitution was made at a position in GTF 7527(GTFJ, SEQ ID NO:65) corresponding to position 607 of SEQ ID NO:62,exchanging a Phe for a Tyr residue. GTF 7527 (SEQ ID NO:65) essentiallyis an N-terminally truncated (signal peptide and variable regionremoved) version of the full-length wild type glucosyltransferase(represented by SEQ ID NO:60) from Streptococcus salivarius (see Table1). Substitutions made in SEQ ID NO:65 can be characterized assubstituting for native amino acid residues, as each amino acidresidue/position of SEQ ID NO:65 (apart from the Met-1 residue of SEQ IDNO:65) corresponds accordingly with an amino acid residue/positionwithin SEQ ID NO:60. In reactions comprising at least sucrose and water,the glucosyltransferase of SEQ ID NO:65 typically produces alpha-glucanhaving about 100% alpha-1,3 linkages and a DP_(w) of 400 or greater(e.g., refer to U.S. Pat. Nos. 8,871,474 and 9,169,506, and U.S. Pat.Appl. Publ. No. 2017/0002336, which are incorporated herein byreference). Glucan synthesis reactions were prepared as follows usingGTF 7527 (SEQ ID NO:65) or a version thereof comprising a Phe-to-Tyrsubstitution at the position corresponding to position 607 of SEQ IDNO:62: vessel, 250-mL indented shake flask agitated at 100 rpm; initialpH, 5.5; reaction volume, 50 mL; sucrose, 100.1 g/L; GTF, 100 U/L;KH₂PO4, 25 mM; temperature, 25° C.; time, 20 hours. The profiles of eachreaction (as measured via methodology similar to that disclosed inExample 1), which were run in duplicate, are provided in Table 4.

TABLE 4 Product Profiles of GTF 7527 (SEQ ID NO: 65) and a Single AminoAcid-Substituted Variant thereof Yield Alpha- Su- Glucan^(d) Olig- crosebased on Leucrose Glucose omer^(c) Fructose GTF Conv. Glucosyl YieldYield Yield balance 7527^(a) 99.7% 29.24% 42% 4.20% 28% 105.62% 752799.8% 22.21% 43% 6.26% 29% 109.02% F607Y^(b) 99.8% 64.92% 16% 3.33% 15%102.73% F607Y 99.8% 62.97% 17% 3.35% 17% 109.17% ^(a)GTF 7527, SEQ IDNO: 65. ^(b)F607Y, version of GTF 7527 (SEQ ID NO: 65) comprising aPhe-to-Tyr substitution at the position corresponding to position 607 ofSEQ ID NO: 62. ^(c)“Oligomer”, gluco-oligosaccharides (believed to allor mostly be of DP ≤ 7 or 8). ^(d)“Alpha-Glucan”, insoluble alpha-1,3glucan.

Based on the data in Table 4, it is apparent that the F607Y substitutionin GTF 7527 (SEQ ID NO:65) can increase this enzyme's yield ofalpha-1,3-glucan and/or decrease its leucrose yield in glucan synthesisreactions, for example.

Ala-510-Glu, Ala-510-Val, or Ala-510-Cys

Example 1 demonstrated, for example, that substitutions in GTF 6855 (SEQID NO:4) at the position corresponding to position 510 of SEQ ID NO:62affected enzyme activity (Table 3). In particular, substitutions of theAla residue with a Glu, Ile, or Val residue all had significant effectson alpha-1,3 glucan yield (increased) and leucrose yield (decreased)compared to the respective yields of the non-substituted enzyme.

To test whether these or similar substitutions could similarly affectyields in different GTFs, substitutions were made at positions in GTFs2919 (SEQ ID NO:28), 0427 (SEQ ID NO:26), 5926 (SEQ ID NO:14), 0847 (SEQID NO:2), 0544 (SEQ ID NO:12), 2379 (SEQ ID NO:6), 5618 (SEQ ID NO:18),4297 (SEQ ID NO:16), 1366 (SEQ ID NO:24), and 6907 (SEQ ID NO:36)corresponding to position 510 of SEQ ID NO:62, exchanging an Ala for aGlu, Val, or Cys residue. Each of these GTFs essentially is anN-terminally truncated (signal peptide and variable region removed)version of a full-length wild type glucosyltransferase (e.g., refer torespective GENBANK annotation information, such as that listed in Table1). Substitutions made in each of SEQ ID NOs:28, 26, 14, 2, 12, 6, 18,16, 24 and 36 can be characterized as substituting for native amino acidresidues, as each amino acid residue/position of these sequences (apartfrom the Met-1 residues of each) corresponds accordingly with an aminoacid residue/position within each respective full-length wild typeglucosyltransferase counterpart. Table 2 lists the alpha-glucantypically produced by each of SEQ ID NOs:28, 26, 14, 2, 12, 6, 18, 16,24 and 36 in reactions comprising at least sucrose and water.

Preparation of GTF 2919 (SEQ ID NO:28), 0427 (SEQ ID NO:26), 5926 (SEQID NO:14), 0847 (SEQ ID NO:2), 0544 (SEQ ID NO:12), 2379 (SEQ ID NO:6),5618 (SEQ ID NO:18), 4297 (SEQ ID NO:16), 1366 (SEQ ID NO:24), or 6907(SEQ ID NO:36), or versions thereof comprising a substitution at theposition corresponding to position 510 of SEQ ID NO:62 was performed asfollows. Codon-optimized (for E. coli) sequences encoding each of theseGTFs were individually cloned into a suitable plasmid for bacterialexpression. Each construct was then transformed into E. coli BL21-AI(Invitrogen, Carlsbad, Calif.). Transformed strains were grown in 10 mLauto-induction medium (10 g/L Tryptone, 5 g/L Yeast Extract, 5 g/L NaCl,50 mM Na₂HPO₄, 50 mM KH₂PO₄, 25 mM (NH₄)₂SO₄, 3 mM MgSO₄, 0.75%glycerol, 0.075% glucose, 0.05% arabinose) containing 100 mg/Lampicillin at 37° C. for 20 hours under 200 rpm agitation. The cellswere harvested by centrifugation at 8000 rpm at 4° C. and resuspended in1 mL of 20 mM sodium phosphate buffer pH 6.0 with CelLytic™ Express(Sigma, St. Louise, Mo.) according to the manufacturer's instructions.In addition, resuspended cells were subjected to no less than onefreeze-thaw cycle to ensure cell lysis. Lysed cells were centrifuged for10 minutes at 12,000 g at room temperature. Each resulting supernatantwas analyzed by SDS-PAGE to confirm expression of the particular GTFenzyme being expressed. Each supernatant was kept on ice at 4° C. untilenzyme activity could be determined (within 1 hour), and/or stored at−20° C.

Glucan synthesis reactions were prepared, and the products thereofanalyzed, largely according to the disclosure of U.S. Pat. Appl. Publ.No. 2014/0087431, which is incorporated herein by reference. Eachreaction was run for 24-30 hours. The profiles of each reaction areprovided in Table 5.

TABLE 5 Product Profiles of Various GTFs and Single AminoAcid-Substituted Variants thereof Yield Alpha- Su- Glucan Olig- crosebased on Leucrose Glucose omer Fructose GTF Conv. Glucosyl Yield YieldYield balance 2919^(a) 92% 20% 28% 15% 37% 90% A510E^(b) 98% 40% 13% 15%31% 93% A510V^(b) 97% 45% 15% 15% 26% 84% A510C^(b) 95% 35% 19% 15% 32%87% 0427^(a) 96% 15% 33% 11% 41% 97% A510E^(b) 96% 1.0%  40% 16% 43%104%  A510V^(b) poor conversion A510C^(b) 96%  9% 30% 12% 50% 97%5926^(a) 97% 12% 37% 11% 41% 93% A510E^(b) 96% 12% 40% 14% 34% 94%A510V^(b) 97% 25% 31% 14% 31% 81% A510C^(b) 97% −1% 35% 14% 52% 97%0847^(a) 97% 18% 33% 11% 38% 92% A510E^(b) 98% 11% 35% 14% 40% 95%A510V^(b) 80% 32% 21% 16% 31% 80% A510C^(b) 97% 10% 33% 13% 44% 97%0544^(a) 99% 37% 22%  8% 33% 86% A510E^(b) 93% 46% 21%  8% 25% 85%A510V^(b) poor conversion A510C^(b) 92% 39% 16%  9% 37% 90% 2379^(a) 95% 4% 30% 18% 48% 92% A510E^(b) 97% −2% 23% 23% 56% 93% A510V^(b) 94%  5%20% 23% 52% 82% A510C^(b) 93% −10%  37% 21% 53% 101%  5618^(a) 99% 80%10%  5%  5% 89% A510E^(b) 94% 82%  5%  4%  9% 93% A510V^(b) 99% 83%  7% 5%  5% 78% A510C^(b) 98% 83%  9%  4%  4% 96% 4297^(a) 97% 78% 12%  6% 4% 86% A510E^(b) 99% 84%  7%  4%  5% 83% A510V^(b) 99% 78%  8%  8%  6%77% A510C^(b) 80% 71%  8%  9%  7% 84% 1366^(a) 97% 12% 39%  7% 43% 91%A510E^(b) 99%  9% 39% 16% 36% 89% A510V^(b) 78% 17% 28% 16% 39% 80%A510C^(b) 97%  1% 39% 12% 48% 96% 6907^(a) 85%  7% 42% 17% 34% 91%A510E^(b) 89% 14% 35% 25% 26% 94% A510V^(b) poor conversion A510C^(b)poor conversion ^(a)GTF 2919 (SEQ ID NO: 28), 0427 (SEQ ID NO: 26), 5926(SEQ ID NO: 14), 0847 (SEQ ID NO: 2), 0544 (SEQ ID NO: 12), 2379 (SEQ IDNO: 6), 5618 (SEQ ID NO: 18), 4297 (SEQ ID NO: 16), 1366 (SEQ ID NO:24), or 6907 (SEQ ID NO: 36). ^(b)A510E/V/C, version of listed GTF(footnote [a]) comprising a substitution with Glu, Val, or Cys at theposition corresponding to position 510 of SEQ ID NO: 62. ^(c)“Oligomer”,gluco-oligosaccharides.

Based on the data in Table 5, it is apparent that some substitutions invarious GTFs at the position corresponding to position 510 of SEQ IDNO:62 can increase a GTF's yield of alpha glucan and/or decrease itsleucrose yield in glucan synthesis reactions, for example.

Example 3 Analysis of the Effects of Two or More Amino AcidSubstitutions on Glucosyltransferase Selectivity Toward Alpha-GlucanSynthesis

This Example describes the effects of introducing multiple amino acidsubstitutions to a glucosyltransferase and determining their effect onenzyme selectivity toward alpha-glucan synthesis.

Briefly, certain amino acid substitutions were made to SEQ ID NO:4 (GTF6855, see Table 1 and Example 1 for description of thisglucosyltransferase). These substitutions are listed in Table 6 below.Each variant enzyme was entered into a glucan synthesis reaction withparameters that were the same as, or similar to, the following: vessel,250-mL indented shake flask agitated at 120 rpm; initial pH, 5.7;reaction volume, 50 mL; sucrose, 75 g/L; GTF, 1.5 mL lysate of E. colicells heterologously expressing enzyme; KH₂PO₄, 20 mM; temperature, 30°C.; time, about 20-24 hours. The alpha-1,3 glucan yield of each reaction(as measured via methodology similar to that disclosed in Example 1) isprovided in Table 6.

TABLE 6 Alpha-1,3 Glucan Yields of GTF 6855 (SEQ ID NO: 4) Variants withMultiple Amino Acid Substitutions Alpha-1,3 GTF^(a) Glucan^(b) Yield^(c)A510D/F607Y/R741S 72.6% A510D/F607Y/N743S 79.2% A510D/F607Y/D948G 88.2%A510D/R741S/D948G 74.5% A510D/F607Y/R741S/D948G 82.8%A510E/F607Y/R741S/R1172C 78.2% A510D/F607Y/D820G/D948G 87.8%A510D/F607Y/D948G/R1172C 88.6% A510D/F607Y/N743S/D948G/R1172C 89.4%A510D/F607Y/R741S/L784Q/F929L/R1172C 79.3% ^(a)Each listed GTF is aversion of GTF 6855 (SEQ ID NO: 4) comprising substitutions atrespective positions, where each position number is in correspondencewith the residue numbering of SEQ ID NO: 62. ^(b)Insoluble alpha-1,3glucan product. ^(c)Alpha-1,3-glucan yield based on glucosyl.

Based on the data in Table 6, it is apparent that introduction ofmultiple amino acid substitutions to GTF 6855 (SEQ ID NO:4) can increasethis enzyme's yield of alpha-1,3-glucan; for example, compare theseyields to those of GTF 6855 (SEQ ID NO:4) without substitutions shown inTable 3. Each of the variant GTF enzymes listed in Table 6 alsoexhibited significant reductions in yields of leucrose, glucose andgluco-oligomers (data not shown).

It is apparent, for example, that a GTF with multiple substitutions suchas at positions corresponding to positions 510 and/or 607 of SEQ IDNO:62 can increase a GTF's yield of alpha glucan.

Example 4 Analysis of the Effects of Additional Amino Acid SubstitutionCombinations on Glucosyltransferase Selectivity Toward Alpha-GlucanSynthesis

This Example describes the effects of introducing multiple amino acidsubstitutions to a glucosyltransferase and determining their effect onenzyme selectivity toward alpha-glucan synthesis. While this analysissupplements the analysis disclosed above in Example 3, it is interestingto note that several of the additional amino acid substitutioncombinations provide modified glucosyltransferases with even higheralpha-1,3-glucan yields.

Briefly, certain combinations of amino acid substitutions were made toSEQ ID NO:4 (GTF 6855, see Table 1 and Example 1 for description of thisglucosyltransferase) by site-directed mutagenesis of appropriate DNAtemplates contained in a plasmid. The plasmid sequences encoding eachmodified glucosyltransferase were individually sequenced to confirm theintended codon changes. Each combination of substitutions is listed inTable 7 below.

Expression plasmids encoding the modified glucosyltransferases wereindividually used to transform a B. subtilis strain containing nineprotease deletions (amyE::xylRPxylAcomK-ermC, degUHy32, oppA,ΔspollE3501, ΔaprE, ΔnprE, Δepr, ΔispA, Δbpr, Δvpr, ΔwprA, Δmpr-ybfJ,ΔnprB). Transformed cells were spread onto LB plates supplemented with 5μg/mL chloramphenicol. Colonies growing on these plates were streakedseveral times onto LB plates with 25 μg/mL chloramphenicol. Eachresulting Bacillus strain for expressing a particular variantglucosyltransferase was then grown for 6-8 hours in LB medium containing25 μg/mL chloramphenicol, and then subcultured into Grants II medium at30° C. for 2-3 days. The cultures were spun at 15000 g for 30 minutes at4° C., and the supernatants were filtered through 0.22-μm filters. Thefiltered supernatants, each of which contained an expressed secretedvariant glucosyltransferase, were aliquoted and frozen at −80° C., andlater used (below) for analyzing alpha-1,3-glucan synthesis activity.

The same amount of each variant enzyme, activity-wise, was entered intoa glucan synthesis reaction with parameters that were the same as, orsimilar to, the following: vessel, 500-mL jacketed reactor withTeflon®-pitched blade turbine (45-degree angle) on a glass stir rod andagitated at 50-200 rpm; initial pH, 5.5; reaction volume, 500 mL;sucrose, 108 g/L; KH₂PO₄, 1 mM; temperature, 39° C.; time, about 18-24hours; filtrate from a previous alpha-1,3-glucan synthesis reaction, 50vol %. The alpha-1,3 glucan yield of each reaction (as measured viamethodology similar to that disclosed in Example 1) is provided in Table7.

TABLE 7 Alpha-1,3 Glucan Yields of GTF 6855 (SEQ ID NO: 4) Variants withMultiple Amino Acid Substitutions Alpha-1,3- Glucan^(b) GTF^(a)Yield^(c) A510D Q588L F607Y R741S D948G R722H T877K M1253I K1277N 88%A510D Q588L F607Y R741S D948G R722H T877K V1188E M1253I Q957P 92% A510DQ588L F607Y R741S D948G T877K V1188E M1253I Q957P 91% A510D Q588L F607YR741S D948G M1253I 89% A510D Q588L F607W R741S D948G 91% Q588L F607YR741S D948G 91% A510D Q588L F607Y R741S D948G N628D T635A T877K M1253IF929L R1172C 92% A510D Q588L F607W R741S D948G S631T S710G R722H T877KV1188E M1253I 94% A510D Q588L F607W R741S D948G S631T S710G R722H T877KV1188E 93% A510D Q588L F607W R741S D948G S631T S710G T877K V1188E M1253I96% A510D Q588L F607Y R741S D948G 89% A510D Q588L F607Y R741S D948GV1188E 88% A510D Q588L F607W R741S D948G S631T S710G V1188E 96% A510DQ588L F607W R741S D948G S710G R722H T877K M1253I 96% A510D Q588L F607YR741S D948G S631T R722H T877K V1188E M1253I 96% A510D Q588L F607W R741SD948G S631T T877K V1188E M1253I 94% A510D Q588L F607W R741S D948G S631TV1188E 98% A510D Q588L F607Y R741S D948G S631T R722H T877K V1188E M1253I95% A510D Q588L F607W R741S D948G V1188E M1253I 93% ^(a)Each listed GTFis a version of GTF 6855 (SEQ ID NO: 4) comprising substitutions atrespective positions, where each position number is in correspondencewith the residue numbering of SEQ ID NO: 62. ^(b)Insoluble alpha-1,3glucan product. ^(c)Alpha-1,3-glucan yield based on glucosyl.

Based on the data in Table 7, it is further apparent that introductionof multiple amino acid substitutions to GTF 6855 (SEQ ID NO:4) canincrease this enzyme's yield of alpha-1,3-glucan; for example, comparethese yields to those of GTF 6855 (SEQ ID NO:4) without substitutionsshown in Table 3. Each of the variant GTF enzymes listed in Table 7 alsoexhibited significant reductions in yields of leucrose, glucose andgluco-oligomers (data not shown).

It is apparent, for example, that a GTF with multiple substitutions,including those at positions corresponding to positions 588, 607 and 741of SEQ ID NO:62, can increase a GTF's yield of alpha glucan.

What is claimed is:
 1. A non-native glucosyltransferase comprising atleast two amino acid substitutions at positions corresponding with aminoacid residues Gln-588, Phe-607, or Arg-741 of SEQ ID NO:62, wherein thenon-native glucosyltransferase synthesizes alpha-glucan comprising1,3-linkages, and wherein the non-native glucosyltransferase has: (i) analpha-glucan yield that is higher than the alpha-glucan yield of asecond glucosyltransferase that only differs from the non-nativeglucosyltransferase at the substitution positions, and/or (ii) aleucrose yield that is lower than the leucrose yield of the secondglucosyltransferase.
 2. The non-native glucosyltransferase of claim 1,wherein the glucosyltransferase comprises amino acid substitutions atpositions corresponding with amino acid residues Gln-588, Phe-607 andArg-741 of SEQ ID NO:62.
 3. The non-native glucosyltransferase of claim1, wherein: (i) the amino acid substitution at the positioncorresponding with amino acid residue Gln-588 is with a Leu, Ala, or Valresidue; (ii) the amino acid substitution at the position correspondingwith amino acid residue Phe-607 is with a Trp, Tyr, or Asn residue;and/or (iii) the amino acid substitution at the position correspondingwith amino acid residue Arg-741 is with a Ser or Thr residue.
 4. Thenon-native glucosyltransferase of claim 1, wherein theglucosyltransferase further comprises at least one amino acidsubstitution at a position corresponding with amino acid residue Ala-510and/or Asp-948 of SEQ ID NO:62; optionally wherein: (i) the amino acidsubstitution at the position corresponding with amino acid residueAla-510 is with an Asp, Glu, Ile, or Val residue; and/or (ii) the aminoacid substitution at the position corresponding with amino acid residueAsp-948 is with a Gly, Val, or Ala residue.
 5. The non-nativeglucosyltransferase of claim 1, wherein the glucosyltransferase furthercomprises at least one amino acid substitution at a positioncorresponding with amino acid residue Ser-631, Ser-710, Arg-722, and/orThr-877 of SEQ ID NO:62; optionally wherein: (i) the amino acidsubstitution at the position corresponding with amino acid residueSer-631 is with a Thr, Asp, Glu, or Arg residue; (ii) the amino acidsubstitution at the position corresponding with amino acid residueSer-710 is with a Gly, Ala, or Val residue; (iii) the amino acidsubstitution at the position corresponding with amino acid residueArg-722 is with a His or Lys residue; and/or (iv) the amino acidsubstitution at the position corresponding with amino acid residueThr-877 is with a Lys, His, or Arg residue.
 6. The non-nativeglucosyltransferase of claim 1, wherein the glucosyltransferase furthercomprises at least one amino acid substitution at a positioncorresponding with amino acid residue Val-1188, Met-1253, and/or Gln-957of SEQ ID NO:62; optionally wherein: (i) the amino acid substitution atthe position corresponding with amino acid residue Val-1188 is with aGlu or Asp residue; (ii) the amino acid substitution at the positioncorresponding with amino acid residue Met-1253 is with an Ile, Leu, Ala,or Val residue; and/or (iii) the amino acid substitution at the positioncorresponding with amino acid residue Gln-957 is with a Pro residue. 7.The non-native glucosyltransferase of claim 1, wherein the alpha-glucanis insoluble and comprises at least about 50% alpha-1,3 linkages, andoptionally wherein the alpha-glucan has a weight average degree ofpolymerization (DP_(w)) of at least
 100. 8. The non-nativeglucosyltransferase of claim 7, comprising a catalytic domain that is atleast about 90% identical to residues 55-960 of SEQ ID NO:4, residues54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20.
 9. The non-nativeglucosyltransferase of claim 8, comprising an amino acid sequence thatis at least about 90% identical to SEQ ID NO:4, SEQ ID NO:65, SEQ IDNO:30, SEQ ID NO:28, or SEQ ID NO:20.
 10. The non-nativeglucosyltransferase of claim 8, wherein the non-nativeglucosyltransferase synthesizes insoluble alpha-1,3-glucan having atleast about 90% alpha-1,3-linkages.
 11. The non-nativeglucosyltransferase of claim 1, wherein the alpha-glucan yield is atleast about 10% higher than the alpha-glucan yield of the secondglucosyltransferase.
 12. A polynucleotide comprising a nucleotidesequence encoding a non-native glucosyltransferase according to claim 1,optionally wherein one or more regulatory sequences are operably linkedto the nucleotide sequence, and preferably wherein said one or moreregulatory sequences include a promoter sequence.
 13. A reactioncomposition comprising water, sucrose, and a non-nativeglucosyltransferase according to claim
 1. 14. A method of producingalpha-glucan comprising: (a) contacting at least water, sucrose, and anon-native glucosyltransferase enzyme according to claim 1, wherebyalpha-glucan is produced; and (b) optionally, isolating the alpha-glucanproduced in step (a).
 15. A method of preparing a polynucleotidesequence encoding a non-native glucosyltransferase, said methodcomprising: (a) identifying a polynucleotide sequence encoding a parentglucosyltransferase that (i) comprises an amino acid sequence that is atleast about 40% identical to SEQ ID NO:4 or positions 55-960 of SEQ IDNO:4, and (ii) synthesizes alpha-glucan comprising 1,3-linkages; and (b)modifying the polynucleotide sequence identified in step (a) tosubstitute at least two amino acids of the parent glucosyltransferase atpositions corresponding with amino acid residues Gln-588, Phe-607, orArg-741 of SEQ ID NO:62, thereby providing a polynucleotide sequenceencoding a non-native glucosyltransferase that has: (i) an alpha-glucanyield that is higher than the alpha-glucan yield of the parentglucosyltransferase, and/or (ii) a leucrose yield that is lower than theleucrose yield of the parent glucosyltransferase.
 16. The method ofclaim 15, wherein said identifying step is performed: (a) in silico, (b)with a method comprising a nucleic acid hybridization step, (c) with amethod comprising a protein sequencing step, and/or (d) with a methodcomprising a protein binding step; and/or wherein said modifying step isperformed: (e) in silico, followed by synthesis of the polynucleotidesequence encoding the non-native glucosyltransferase enzyme, or (f)using a physical copy of the polynucleotide sequence encoding the parentglucosyltransferase.